An ancestral role for Semaphorin3F-Neuropilin signalling in patterning neural crest within the new

48 Pages·2017·3.92 MB·English

by  Joshua Ryan York

©2018.PublishedbyTheCompanyofBiologistsLtd|Development(2018)145,dev164780.doi:10.1242/dev.164780
RESEARCHARTICLE
An ancestral role for Semaphorin3F-Neuropilin signaling in
patterning neural crest within the new vertebrate head
JoshuaR.York,TianYuan,OlgaLakizaandDavidW.McCauley*
ABSTRACT Much of the head skeleton and sensory organ systems of
vertebrates are formed during embryonic development from the
Theoriginofthevertebrateheadisoneofthegreatunresolvedissues
neuralcrest,amigratoryandmultipotentcellpopulationuniqueto
invertebrateevolutionarydevelopmentalbiology.Althoughmanyof
the vertebrate lineage (Donoghue et al., 2008; Hall, 2008; His,
the novelties in the vertebrate head and pharynx derive from
1868;LeDouarin,1999;MuñozandTrainor,2015;Santagatiand
the neural crest, it is still unknown howearly vertebrates patterned
Rijli,2003).Atthemolecularlevel,neuralcrestcelldevelopmentis
the neural crest within the ancestral body plan they inherited from
controlledbyacomplex,integratedgeneregulatorynetwork(GRN)
invertebrate chordates. Here, using a basal vertebrate, the sea
that progressively refines the developmental state of this cell type
lamprey,weshowthathomologsofSemaphorin3F(Sema3F)ligand
from early induction and specification to terminal differentiation
anditsNeuropilin(Nrp)receptorsshowcomplementaryanddynamic
(Betancur et al., 2010; Meulemans and Bronner-Fraser, 2004;
patternsofexpressionthatcorrelatewithkeyperiodsofneuralcrest
Sauka-Spengler and Bronner-Fraser, 2008; Simões-Costa and
development (migration and patterning of cranial neural crest-
Bronner, 2015). Under the control of this GRN, neural crest cells
derived structures). Using CRISPR/Cas9-mediated mutagenesis,
are specified in the dorsal-most part of the embryonic central
wedemonstratethatlampreySema3Fisessentialforpatterningof
nervous system, from which they detach and then migrate
neural crest-derived melanocytes, cranial ganglia and the head
throughout the head and trunk (Bronner, 2012; Clay and
skeleton,butisnotrequiredforneuralcrestmigrationorpatterningof
Halloran, 2010; Duband et al., 1995). After arriving at their
trunk neural crest derivatives. Based on comparisons with jawed
destinations, neural crest cells differentiate into an array of cell
vertebrates,ourresultssuggestthatthedeploymentofNrp-Sema3F
types, including melanocytes, smooth muscle cells, neurons and
signaling, along with other intercellular guidance cues, was pivotal
glia of the peripheral sensory nervous system, as well as cartilage
in allowing early vertebratesto organize and pattern cranial neural
and bone that comprise the head and pharyngeal skeleton (Green
crest cells into many of the hallmark structures that define the
etal.,2015).
vertebratehead.
Although neural crest cells are a vertebrate innovation, the
KEYWORDS:Lamprey,Pharynx,Cartilage,Cranialganglia,Head pharyngeal apparatus, where the neural crest builds much of the
skeleton,Craniofacialdevelopment,Vertebrateevolution headskeletonandsensorysystems,isnot.Infact,thepharynxisa
generalfeatureofdeuterostomeembryos,asisthegeneregulatory
network that orchestrates pharyngeal development (Gillis et al.,
INTRODUCTION 2012;Ouetal.,2012;Rycheletal.,2005;Veitchetal.,1999).Thus,
Akeyeventinearlyvertebrateevolutionwasthetransitionfroma theformationofthenewvertebrateheadrequirednotonlytheorigin
sessile,filter-feedinglifestyletooneofactivepredation(Gansand of new cell types and gene regulatory networks, but also the
Northcutt, 1983). This event was driven, in large part, by a integration and coordination of ancestral (pharynx development)
transformation of the ancestral chordate pharynx, resulting in and derived (neural crest development) developmental-genetic
the vertebrate ‘new head’ (Forey and Janvier, 1994; Gans and programs (Graham and Richardson, 2012; Veitch et al., 1999).
Northcutt,1983;Northcutt,2005;NorthcuttandGans,1983).The Despitethesignificanceofthiseventinearlyvertebrateevolution,
newvertebratepharynxwasmuscularizedandbuttressedbyarobust the molecular mechanisms that coupled these two developmental
endoskeleton made of cellular cartilage, which in turn provided processesareunknown.
support and protection for a complex central nervous system and In jawed (gnathostome) vertebrates, the migratory routes of
pairedsensoryorgans,therebyfacilitatingamoreactive,predatory neural crest cells and patterning of neural crest-derived structures
lifestyle (Donoghue and Keating, 2014; Graham, 2001; Square in the head and pharynx are controlled, in part, by signaling
etal.,2016b).Itwastheintegrationandcoordinationofthesetraits interactions between receptors on neural crest cells and their
that distinguished the first vertebrates morphologically and correspondingligandssecretedfromothercellsintotheextracellular
behaviorallyfromtheirclosestrelatives:theinvertebratechordates environment (Gammill et al., 2007, 2006; Krull et al., 1997;
(DonoghueandKeating,2014;GansandNorthcutt,1983). Minoux and Rijli, 2010). Examples of such signaling systems
includeRobo-Slit,Eph-Ephrin,CXCR4-Sdf,andNeuropilin(Nrp)/ T
N
Plexin-Semaphorin(Sema),eachofwhichpatternsneuralcrestcells
E
by attraction, repulsion, or acombination thereof (Theveneau and M
DepartmentofBiology,UniversityofOklahoma,730VanVleetOval,Norman,OK,
Mayor,2012,2014).Althoughmanyofthesesignalingsystemsare
73019,USA. P
evolutionarilyconservedbetweenvertebratesandinvertebrates,one O
*Authorforcorrespondence([email protected]) particulargroup,theclassIIIfamilyofSemaligands(Sema3)and L
E
O.L.,0000-0003-4499-3289;D.W.M.,0000-0002-4190-9304 theirNrpreceptors(Nrp1andNrp2),emergedandwereduplicated V
withinthevertebratelineage(YazdaniandTerman,2006).Thus,the E
Received19February2018;Accepted27June2018 deployment of Nrp/Sema3 signaling in vertebrates correlates with D
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RESEARCHARTICLE Development(2018)145,dev164780.doi:10.1242/dev.164780
the appearance of neural crest cells and the new vertebrate head, RESULTS
suggesting a link between Nrp/Sema3-mediated neural crest MolecularphylogeneticsofvertebrateclassIIISemaphorins
patterning and the origin of vertebrate novelties, such asthe head andNeuropilins
skeleton and sensoryorgan systems. This hypothesis is supported Previous research identified a Class III (Sema3) and a Class IV
bythefactthat,injawedvertebrates,Sema3(Sema3DandSema3F) (Sema4)Semaphorin,aswellasSemareceptors(Nrps)inlamprey
andNrp(Nrp1andNrp2)proteinactivityisnecessarytoorganize (Shifman and Selzer, 2006, 2007). To investigate Nrp/Sema3
migratory neural crest and pattern neural crest-derived cranial signaling activity during neural crest development in lamprey, we
sensorygangliaandelementsoftheheadandpharyngealskeleton PCRamplifiedandcloneda553-bpsequenceofthegenepreviously
(Berndt and Halloran, 2006; Gammill et al., 2007, 2006; Kulesa identified as encoding Sema3. Our analysis of the sea lamprey
and Gammill, 2010; Yu and Moens, 2005). However, it is genome uncovered additional Sema3s (Fig. S1A). Phylogenetic
unknown whether a key Nrp/Sema3 patterning function for analysisconfirmedwithstrongsupportthattheSema3sequencefirst
neuralcrestisuniquetothejawedvertebrateclade,orisinsteada identified (Shifman and Selzer, 2006, 2007) is a member of the
deeply conserved feature of neural crest biology that was also Sema3Fclade(Fig.S1A)andalsoconfirmedthatanotherpreviously
presentinthefirstjawless(agnathan)vertebratesover500million identifiedSema3inlampreyislikelyamemberoftheSema3Dclade
yearsago. (Fig.S1A)(Sauka-Spengleretal.,2007).Ourgenomicsearchesalso
To distinguish between these possibilities, we examined the uncovered three lamprey homologs of vertebrate Nrp1 and Nrp2
expression patterns of the Nrp/Sema3F signaling system, and the receptors.Ourphylogeneticanalysissuggestedthat,similartojawed
functionalrolesofSema3Fproteinduringneuralcrestdevelopment vertebrates, lampreys have two Nrp paralogy groups (Fig. S1B).
in a basal vertebrate, the sea lamprey (Petromyzon marinus). However, our analysis was unable to resolve strict paralogy of
Lampreysareagroupofjawlessvertebratesthat,alongwithhagfish, lampreyNrpswitheitherjawedvertebrateNrp1orNrp2.Therefore,
constitutetheonlyextantmembersofthecyclostome(‘agnathan’) we named these groups NrpA and NrpB (Fig. S1B), with the
clade, which includes diverse fish-like forms that first appeared two NrpA copies (NrpA1 and NrpA2) likely originating from a
duringthePaleozoicera(McCauleyetal.,2015)andarethesister lamprey-specificgeneduplicationevent(Fig.S1B).
taxaofallotherlivingvertebrates.Giventhattheyoccupythemost
basal phylogenetic position among extant vertebrates, and readily LampreyNrp-Sema3Fexpressioncorrelateswithpatterning
produce embryos that are amenable to experimental analysis, ofcranialneuralcrest
lampreysareidealevolutionaryanddevelopmentalmodelstostudy As a first step toward understanding the contribution of Nrp/
the origin of vertebrate-specific traits (Green and Bronner, 2014; Sema3Fsignalingtoneuralcrestdevelopmentinbasalvertebrates,
McCauley et al., 2015) through comparisons with developmental we characterized their expression patterns throughout lamprey
mechanismsinjawedvertebrates.Lampreys,similartotheirjawed embryogenesis. In jawed vertebrates, early Sema3F expression
vertebraterelatives,haveneuralcrestcellsthatmigrateintothehead occursduringneuralcrestmigration,oftenlocalizingtoneuralcrest-
andpharynx,followingstereotypedroutes(McCauleyandBronner- freezonesoftheforebrain,hindbrainandpharynx,withmigratory
Fraser,2003;Yorketal.,2017).Onceattheirtargeteddestinations, neuralcrestshowingcomplementaryexpressionofNrp1/2receptors
these neural crest cells are patterned into a pharyngeal and head (Gammilletal.,2007).Wefoundcomparableexpressionpatternsof
skeleton made of cellular cartilage, as well as cranial sensory Sema3F/Nrpsduringlampreyneuralcrestdevelopment (Figs1-7).
ganglia (Jandzik et al., 2014; Lakiza et al., 2011; McCauley and In Tahara stage (T) 22 lamprey embryos, neural crest cells are
Bronner-Fraser,2003,2006;Squareetal.,2016b).Althoughthese migrating(Sauka-Spengleretal.,2007;Squareetal.,2016a;Tahara,
structures arepresumed to behomologousto cranialcartilage and 1988; York et al., 2017). At this time, Sema3F transcripts were
gangliainjawedvertebrates(McCauleyandBronner-Fraser,2006; enriched in forebrain and hindbrain, with expression appearing in
Modrell et al., 2014), the molecular, cellular and genetic the ectoderm and evaginating endodermal pouch near pharyngeal
mechanisms responsible for their patterning in jawless vertebrates arch(pa)3(Fig.1A,B).ByT23,Sema3Fexpressionweakensinthe
areunknown. forebrain concomitant with expansion into pa3-4 (Fig. 1C), with
Ourresults,togetherwiththeprioridentificationoflampreyNrp expressioninthenascentendodermalpouchofpa4(Fig.1C,D).At
andSema3genes(Sauka-Spengleretal.,2007;ShifmanandSelzer, earlyT24,postmigratorycrestcellshavecolonizedthepharynxand,
2006,2007)revealthatthelampreygenome,similartothatofmany by late T24, gradually become restricted within each of the
other vertebrates, encodes homologs of both Nrp1/2 and Sema3. differentiating pharyngeal arches (McCauley and Bronner-Fraser,
FocusingonNrp/Sema3Fsignaling,weshowthatSema3Fandits 2003).Ourexpressionanalysisatthesestagesshowedthatexpanded
Nrpreceptors(lampreyNrpA,NrpBandNrpC)showdynamicand Sema3F expression accompanied caudal differentiation of pa3-6,
complementarypatternsofexpressionthatcorrelatewithkeysteps with expression gradually increasing within the pharyngeal
ofneuralcrestdevelopment(migration;earlyandlatepatterningof ectoderm along the anteroposterior axis (pa3-5 in Fig. 1E,F, and
pigment, cranial sensory neurons and head skeleton), similar to pa3-6inFig.1G,H).
jawed vertebrates. Using clustered regularly interspaced short During T25/26,thelampreypharynx elongates andneuralcrest
palindromic repeat (CRISPR)/Cas9-mediated genome editing, we cellswithineachpagraduallycoalesceintodorsal-ventralstacksof
demonstrate that Sema3F signaling is not required for the prechondrocytesthatprefigurethelarvalcartilagebarswithinpa3-9 T
N
segregation of migratory neural crest streams, but is essential for (Martin et al., 2009; McCauley and Bronner-Fraser, 2003, 2006).
E
patterningofpigment,cranialsensoryneuronsandelementsofthe Sema3Fshoweddynamicexpressionduringtheseearlypatterning M
head and pharyngeal skeleton at multiple stages of development. events of the head skeleton (Fig. 2). Continuing from pharyngeal P
Takentogether,ourresultssuggestthatNrpreceptorsandSema3,as ectodermal expression at late T24 (Fig. 1G,H), by T25, Sema3F O
avertebrate-specificsignalingsystem,actascellularguidancecues mRNA expands into pa1-7 (Fig. 2A), with expression in the L
E
topatternthecranialneuralcrestintovertebrate-specificnovelties, pharyngealectoderm(Fig.2B)andepitheliumliningtheoralcavity V
whichallowedstemvertebratestocoordinateneuralcrestmigration (Fig. 2C). The relatively broad ectodermal Sema3F expression E
D
anddifferentiationprograms. accompanying the pharyngeal arches at T25 gave way to sharp
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RESEARCHARTICLE Development(2018)145,dev164780.doi:10.1242/dev.164780
paralogswereexpressedinmigratorycranialneuralcrestcolonizing
the mouth and pa1-3, and in the cranial ectoderm (Fig. 3A-D).
Similarly, from T23 to late T24, late-migrating and postmigratory
neural crest cells expressing NrpA1 and NrpA2 filled the lateral
marginwithinthemesenchymalcoreofeachofthedifferentiating
pharyngealarches(Fig.3E-P).NrpBexpressionwassimilartothat
ofNrpA1andNrpA2inmigratorycranialneuralcrestenteringthe
anteriororopharynxandmoreposteriorpharyngealarchesfromT22
to early T24 (Fig. 4A-F), eventuallyoccupying the mesenchymal
coreofeacharchbylateT24(Fig.4G,H).
ContinuingfromlateT24,NrpA1-positiveneuralcrestatT25still
occupiedthelateralmesenchymalcoreofeachofpharyngealarch
(Fig. 5A,B) and the oral mesenchyme (Fig. 5C). By T26, some
NrpA1-expressing neural crest in the lateral mesenchymal core
began to coalesce into the characteristic circular shape of
pharyngeal prechondrocytes (Fig. 5D,E; compare Fig. 2F), and
was expressed strongly in presumptive mucocartilage occupying
pa1andthelateralvelarskeleton(Fig.5F).FromT27toT30,NrpA1
expressionintheheadskeletonwasweakened,withupregulationin
otherpharyngealstructures,includinggillepithelium(Fig.5G-N).
Compared with NrpA1, the NrpA2 paralog showed comparable
patternsofexpressioninpostmigratoryneuralcrestthroughoutthe
head and pharynx at T25 and T26 (Fig. 6A-F), but was also
expressedinthemesodermwithineacharch(Fig.6B,E).FromT27
to T30, NrpA2 mRNAwas lost from pharyngeal prechondrocytes
Fig.1.EarlycranialexpressionoflampreySema3F.(A,B)AtT22,Sema3F (Fig. 6G-J), concomitant with upregulation in the ventral somitic
transcriptslocalizetotheforebrain(fb),hindbrain(hb)andpa3inthe andepibranchialmesoderm(Fig.6H-J),aswellastheneuralcrest-
evaginatingendoderm(arrowhead,B)andectoderm(ect,B).(C,D)This derivedhypobranchialbars(Fig.6I,J)andlarvalgills(Fig.6K-N).
endodermalexpression(arrowheads,D)patternappearsagainatT23inpa3-
LampreyNrpBexpressionduringlaterstagesofheaddevelopment
4,asforebrainexpressionweakens(asterisk,C).(E-H),ThroughoutT24,asits
was similar to that of NrpA1, with transcripts localizing to
expressionstillweakensintheforebrain(comparefbinAwithasterisksinC,E,
G),Sema3Fexpressionexpandsthroughoutthepharyngealectoderm(ect,F, postmigratory crest cells in the mouth and lateral margin of the
H)tomoreposteriorpharyngealarches(pa3-6inE,G).Theanteriorsideis coreofeachpharyngealarchatT25(Fig.7A-C),withasubsetof
facingleftinallpanels.Dashedlinesindicateplaneofsectionshowninthe these cells in each arch contributing to nascent pharyngeal
indicatedpanels.Scalebars:100μm. prechondrocytes at T26 (Fig. 7D,E), as well as elements of the
velar skeleton, pa1 and pa2 (Fig. 7F). In late embryos and early
expressionboundarieswithinpa1-9byT26(Fig.2D).Sectioningof larvae (T27-late T30), NrpB expression was maintained in the
theT26embryoinFig.2DrevealedupregulatedSema3Fexpression oropharynxandupperlip(Fig.7G,H,K,N),whereasexpressionin
intherostralendodermofeachpharyngealpouch,withexpression the neural crest-derived pharyngeal cartilage bars in the posterior
weakeningintheectoderm(Fig.2E,arrowheadsandasterisk).This pharynxfromearlierstageswasreplacedbymesodermalexpression
Sema3Fexpressionpatternoccursjustasneuralcrestcellsinpa3-9 in the ventral somites (Fig. 7G-J) as well as epibranchial
are patterned into SoxE1-positive dorsoventrally stacked rods of mesodermal mesenchyme that contributes to pharyngeal muscle
pharyngeal prechondrocytes (Cattell et al., 2011; McCauley and fibers(Fig.7K-N).
Bronner-Fraser, 2006). Double in situ hybridization for Sema3F In summary, our expression analysis of lamprey Sema3F and
and SoxE1 revealed Sema3F mRNA adjacent to SoxE1-positive NrpsshowedthatlampreyNrpreceptorsoccuronmigratorycranial
prechondrocytesinthepharyngealarches,suggestingthatSema3F neural crest cells colonizing the early embryonic head (T22-T24)
expressionpatternstheearlylampreyheadskeleton(Fig.2F). and maintain expression in postmigratory neural crest that will be
Downregulation of Sema3F in pa3-9 by T27 (Fig. 2G) was patternedintoelementsoftheheadskeleton(T25-T26).Atthesame
followedbyexpressionaroundtheventralmucocartilage,anelastin- time,complementaryexpressionofSema3Fligandemanatesfrom
like cartilage made of mesenchymal chondrocytes embedded in a theadjacentpharyngealectodermandendoderm,inpatternsthatare
looseextracellularmatrix(mc,Fig.2G).Mucocartilageisspecificto spatially and temporally dynamic. From late embryonic stages
jawless vertebrates and is the primary cartilage type in the velum (T27-T28) into early larvaldevelopment (T28-T30), Nrp/Sema3F
(pa1-2), upper and lower lips, and floor of the pharynx (Johnels, expression is gradually lostfrom much of theneural crest-derived
1948;Martinetal.,2009;Yaoetal.,2011).FromT28tolateT30 pharyngeal cartilage bars and is upregulated in cranial mesoderm
Sema3Fexpressionexpandedthroughoutthemucocartilageofthe andmucocartilage. T
N
pharyngealfloor,pa1-2andupperandlowerlips(Fig.2H-N).
E
After determining Sema3F expression in the lamprey head, we LampreySema3Fisnotrequiredformigrationofcranial M
next characterized expression of the corresponding Nrp receptors neuralcrest P
toidentifycelltypesthatmightrespondtoSema3Fsignaling.Our OurresultsshowedthatpharyngealexpressionofNrpsandSema3F O
expression analysis revealed that lamprey, similar to jawed started during neural crest migration and early colonization of the L
E
vertebrates, expresses Nrp orthologs in cranial neural crest pharynx(∼T23-T24,Figs1,3,4).Thiswasfollowedbypharyngeal V
throughout head development in a pattern complementary to that arch expression that is suggestive of a role in patterning cartilage E
of Sema3F expression (Figs 3-7). At T22, NrpA1 and NrpA2 bars of the head skeleton in pa3-9 (∼T25-T26, Figs 2A-F, 5A-F, D
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RESEARCHARTICLE Development(2018)145,dev164780.doi:10.1242/dev.164780
Fig.2.CranialexpressionofSema3Fduringheadskeleton
patterning.(A-C)AtT25,Sema3Fexpressionissegmentalinthe
ectodermofpa1-7(A;ect,B)andinthestomadealepithelium(stinC).
(D-F)ByT26,Sema3Fexpressioninpa1-9(D)resolvessharplytothe
anteriorpharyngealendoderm(arrowheads,E,F),withweakened
ectodermalexpression(asterisk,E),apatternadjacenttoneuralcrest-
derivedpharyngealprechondrocytesexpressingSoxE1(arrows,F).
(G)ByT27,Sema3Fexpressionisupregulatedventrallyin
mucocartilage(mc,arrow).(H-N)AtT28,Sema3Fexpressionremainsin
mucocartilage(mc,H,J),islostfromthepharyngealcartilagebars(cb,J)
andappearsinmesenchymearoundmucocartilageofpa1-2andupper
(ul)andlowerlips(ll),apatterncontinuingthroughT30(K-N).Exceptfor
cross-sectionsinI,J,M,N,allpanelsareorientedwiththeanteriorfacing
left.da,dorsalaorta.DashedlinesinA,D,H,Lindicateplaneofsection
shownintheindicatedpanels.BoxedareasinIandMareshownat
highermagnificationinJandN,respectively.Scalebars:100μm.
6A-F and 7A-F). Finally, Sema3F and Nrp expression occurs in Sema3Fduringneuralcrestdevelopment,weusedCRISPR/Cas9-
mucocartilage, (i.e. pa1-2, floor of the pharynx) and the mediated genome editing, as described in lamprey (Square et al.,
hypobranchial bars of the pharynx (∼T27-30, Figs 2G-N and 2015;Yorketal.,2017;Zuetal.,2016).
6H-J).GiventhatspatiotemporaldifferencesinSema3Fexpression In jawed vertebrates, migratory neural crest cells express
parallel early colonization of the pharynx by Nrp-positive neural transcription factors, such as Sox10 and n-Myc, among others,
crest, formation of cartilage bars in pa3-9, and formation of and are segregated into three migratory streams (Sauka-Spengler
mucocartilage in pa1-2, we asked whether Sema3F signaling is and Bronner-Fraser, 2006, 2008; Wakamatsu et al., 1997). The
required foreach of these processes. To test thefunctional role of division of these streams is enforced in part by repellent Sema3F
T
N
E
M
P
O
Fig.3.EarlycranialexpressionoflampreyNrpA1andNrpA2.AtT22(A-D),NrpA1andNrpA2areexpressedinpharyngealectoderm(ect)and L
migratorycranialneuralcrestcolonizingpa1-3(neuralcrestindicatedbyarrowheads),apatternthatcontinuesintolateT24(E-P)asNrpA1-positiveand E
NrpA2-positiveneuralcrestcellscolonizepa1-6.BylateT24(M-P),bothNrpA1andNrpA2expressionoccupiespharyngealectoderm(ect)andthe V
mesenchymalcoreofeacharch.Allpanelsareorientedwiththeanteriorfacingleft.Dashedlinesindicateplaneofsectionshownintheindicatedpanels. E
D
Scalebars:100μm.
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RESEARCHARTICLE Development(2018)145,dev164780.doi:10.1242/dev.164780
Fig.4.EarlycranialexpressionoflampreyNrpB.(A,B)Migratorycranial
neuralcrestexpressingNrpBentersthepharynxinpa1-3(A),colonizingthe
mesenchymalcoreofnascentpharyngealarches(arrowheads,B).NrpB Fig.5.CranialexpressionofNrpA1duringheadskeletonpatterning.(A-C)
expressionalsooccursinpharyngealectoderm(ect,B).(C-H)Thispatternof AtT25,NrpA1expressionisinpa1-7(A)inthelateralmesenchymalcores
NrpB-positiveneuralcrestmigrationcontinuesinpa1-6fromT23tolateT24 (arrowhead,B),andinupperlip(ul)mesenchymearoundthestomodeum(st,
(neuralcrestindicatedbyarrowheads),butwithweakeningectodermal C).(D-F)T26NrpA1expressionremainsinthelateralmesenchymalcoresof
expression(ect).Allpanelsorientedwiththeanteriorfacingleft.Dashedlines pa1-9(D;arrowhead,E),withsomecellscoalescingintopharyngeal
indicateplaneofsectionshownintheindicatedpanels.Scalebars:100μm. prechondrocytebars(arrows,E),aswellastheskeletonofpa1(F)andlateral
velarskeleton(vs,F).(G-N)AtT27,T28NrpA1pharyngealexpression
signalinginneuralcrest-freezonesofthehead,andfunctionalloss (arrowheads)weakensinthecartilagebars(cb,outlinedinJ),andis
upregulatedinthegills(ginJ),apatternmaintainedintolateT30(K-N).Except
ofSema3Factivityresultsintheirinappropriatemixing,whichcan
forcross-sectionsinI,J,M,N,allpanelsareorientedwiththeanteriorfacingleft,
lead to abnormal patterning of neural crest-derived structures
andthedorsalsideorientedup.Dashedlinesindicateplaneofsectionshownin
(Gammill et al., 2006, 2007; Kulesa et al., 2010; Kulesa and theindicatedpanels.BoxedareasinIandMareshownathighermagnification
Gammill, 2010). Similar to jawed vertebrates, lamprey cranial inJandN,respectively.nt,neuraltube;s,somite.Scalebars:100μm.
neuralcrestcellsmigrateinthreestreamsandexpresshomologsof
Sox10(lampreySoxE2)andnMyc(Lakizaetal.,2011;McCauley (Fig.9C,D).Vertebratecranialsensoryganglia,includingthoseof
andBronner-Fraser,2003;Sauka-Spengleretal.,2007),suggesting lamprey,arethoughttoderivefrombothneuralcrestandectodermal
that Sema3F also functions in lamprey to segregate migratory placode cell populations (Modrell et al., 2014; Schlosser, 2005).
cranial neural crest. However, in contrast to jawed vertebrates, we Giventhis,andbasedonectodermalexpressionofSema3FandNrp
found that, in Sema3F mutant embryos (n=20/20), nMyc+ and genes in lamprey (Figs 1-7), we investigated the gene expression
SoxE2+ neural crest cells still migrated in three distinct streams profilesoftheplacode-specificcranialganglionmarkersPax3/7and
(Fig. 8B,D). These migratory patterns were similar to those in Six1/2, which mark the ophthalmic portion of the trigeminal
negative control embryos (Fig. 8A,C), suggesting the lack of a ganglion (OpV; Pax3/7) and petrosal and posterior lateral line
prominent role for Sema3F in regulating neural crest migration ganglia (pet, pLGG; Six1/2), respectively (Modrell et al., 2014;
during early development. See Fig. S3 for individual Sema3F Schlosser, 2005; Zou et al., 2004). Our results showed that OpV
mutantgenotypesequences. ganglia in Sema3F mutants appeared smaller, with reduced
expression of Pax3/7 (Fig. S2A,B). Moreover, Six1/2-positive
Sema3Fsignalingisessentialforearlypatterningofcranial pLLGandpetgangliacouldnotbediscriminatedfromeachotherin
neuralcrestderivatives mutants, suggesting that they formed as a single fused ganglion
Despite unperturbed patterning of neural crest migration at stage (Fig. S2C,D). These results suggest that proper patterning of
T22-T23 in Sema3F CRISPR mutants (Fig. 8B,D), we observed nonectomesenchymal cranial neural crest derivatives, as well as T
N
inappropriatepatterningofcranialneuralcrestderivativesinolder cranialsensoryplacodes,isdependentonSema3F/Nrpsignaling.
E
mutantembryos(Figs9,10;seeFig.S3formutantgenotypes).At Next,wefocusedonthepossiblepatterningfunctionsofSema3F/ M
T26, we observed differentiated neural crest-derived melanocytes Nrp signaling in neural crest cells during early head skeleton P
in mutants, but these embryos (n=8/10) failed to properly development in lamprey. The SoxE and Twist families of O
position melanocytes in the anterior head and along the dorsal transcription factors are not only widely recognized for L
E
pharynx (Fig. 9A,B). Mutant embryos (n=7/10) also had severely controlling specification of neural crest cells, but are also known V
disorganized cranial ganglia, with apparent fusion and/or to govern patterning of neural crest-derived elements of the head E
D
uncondensed ganglionic neurons compared with controls skeletoninvertebrateembryos(Carletal.,1999;Cattelletal.,2011;
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RESEARCHARTICLE Development(2018)145,dev164780.doi:10.1242/dev.164780
Fig.6.CranialexpressionofNrpA2duringheadskeletonpatterning.
(A-C)AtT25,NrpA2isexpressedinpostmigratoryneuralcrestinpa1-7(A)
inthelateralmesenchymalcores(arrowhead,B)andmesoderm(mesinB), Fig.7.CranialexpressionofNrpBduringheadskeletonpatterning.(A-
andalsoinupperlip(ul)mesenchymearoundthestomodeum(st,C).(D-F) C)NrpBatT25isexpressedinpostmigratoryneuralcrestinpa1-7(A)within
T26NrpA2transcriptsoccurthroughoutthemesenchymalcoresofpa1-9 thelateralcompartmentofthemesenchymalcores(arrowhead,B)andin
(D)intheneuralcrest(arrowhead,E)andmesoderm(mes,E),andin theupperlip(ul)mesenchymecircumscribingthestomodeum(stinC).(D-
prechondrocytesinpa1-2(F),butisabsentfromthevelarskeleton(vs,F). F)T26NrpBexpressionremainsinthelateralmesenchymalcoresofpa1-9
(G-J)AtT27,T28NrpA2expressionisintheupperlip(ul),butinthe (D;arrowhead,E),withsomecellscoalescingintothecircularshapeof
pharynx(arrowheads),expressionweakensinthepharyngealcartilage pharyngealprechondrocytebars(arrows,E),skeletonofpa1-2(F),andthe
bars(cb,outlinedinJ),andisupregulatedinthesomiticandepibranchial velarskeleton(vs,F).(G-J)T27andT28NrpBexpressionremainsinthe
mesoderm,andthehypobranchialbar(ebm,hbbinJ).(K-N)Throughout upperlip(ul),isgraduallylostinthepharynx(arrowheads),frompharyngeal
T30,upperlip(ul)expressionismaintained,withpharyngealexpressionin cartilagebars(cbinJ),butisupregulatedinventralsomites(sinJ),
thegills(ginN).Exceptforcross-sectionsinI,J,M,N,allpanelsareoriented epibranchialmesoderm(ebminJ)andpharyngealarchmuscle(paminJ).
withtheanteriorfacingleftanddorsalsideorientedup.DashedlinesinA,D, (K-N)ThroughoutT30,NrpBexpressionisintheupperlip(ul)andventral
H,Lindicateplaneofsectionshownintheindicatedpanels.BoxedareasinI somite-derivedepibranchialandpharyngealarchmuscle(ebmandpamin
andMareshownathighermagnificationinJandN,respectively.Scale M).nt,neuraltube.Exceptforcross-sectionsinI,J,LandM,allpanelsare
bars:100μm. withtheanteriorfacingleftandthedorsalsideorientedup.Dashedlines
indicateplaneofsectionshownintheindicatedpanels.BoxedareasinIand
LareshownathighermagnificationinJandM,respectively.Scalebars:
Cheung and Briscoe, 2003; McCauley, 2008; Soo et al., 2002). 100μm.
Similar to jawed vertebrates, lamprey homologs of these genes
(SoxE1, SoxE3 and TwistA) are also expressed during early
development of the head skeleton and are required for contact theectoderm laterally (asterisks in Fig.10J-L, compareto
development of cellular cartilage (T25-T26) (Lakiza et al., 2011; Fig.10D-F),leadingtoSoxE1,SoxE3andTwistA-expressingcells
McCauleyandBronner-Fraser,2006;Sauka-Spengleretal.,2007). being able to cross pharyngeal arch boundaries along the
In particular, SoxE1 expression largely occurs in pharyngeal anteroposterior axis (arrowheads, Fig. 10J-L), a result similar to
prechondrocytes in pa3-9 (Cattell et al., 2011; McCauley and that of previous work in lamprey suggesting that correct
Bronner-Fraser,2006),whereasSoxE3andTwistAtranscriptsmark chondrogenesis and patterning of cartilage precursors is
prechondrocytes in all pas, including mucocartilage elements in dependent on proper pharyngeal pouch formation (Jandzik et al.,
pa1-2 and upper and lower lips (McCauley and Bronner-Fraser, 2014). However, even in pharyngeal arches that had outpocketed
2006; Sauka-Spengler et al., 2007; Zhang et al., 2006) (see also completelyornearlyso,westillobservedpostmigratoryneuralcrest
Fig.10A-F).Asexpectedfromourresultsabove(Fig.8),wefound cells that had failed to completely condense into pharyngeal T
N
thatCRISPRtargetingofSema3FdidnotpreventSoxE1+,SoxE3+ prechondrocytes with sharp boundaries (arrows in Fig. 10J,K,L),
E
or TwistA+ cells from migrating into the pharynx (Fig. 10). suggestingthatproperpatterningofpharyngealprechondrocytesin M
However,thecellsexpressingthesegenesdidnotbecomeorganized Sema3Fmutantsinvolvesnotonlyanindirecteffectonpharyngeal P
intoseriallyrepeatingstacksofprechondrocytesinpa3-9compared pouch morphogenesis, but also a specific neural crest patterning O
withcontrolembryos(SoxE1,n=18/20,Fig.10A,G;SoxE3,n=16/ function upon proper pouch formation. We also observed that L
E
20,Fig.10B,H;TwistA,n=15/20;Fig.10C,I).Sectioningofthese Sema3Fmutantembryoshadpatterningdefectsinpa1-2,whichwill V
Sema3F CRISPR mutants revealed that, in some cases, the form the mucocartilage-based elements of the mouth and velar E
D
pharyngealendodermhadfailedtoevaginateproperlyanddidnot skeleton. We observed failure of proper mouth development in
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RESEARCHARTICLE Development(2018)145,dev164780.doi:10.1242/dev.164780
prechondrocytes during earlier embryonic stages in Sema3F
CRISPR mutants (T25-T26, Figs 9,10), we asked whether these
phenotypes persistedinto ammocoete larval stages (T30), or were
correctedlaterindevelopmentbyacompensatorymechanism.We
foundthat,comparedwithcontrollarvae,CRISPR/Cas9-mediated
knockoutofSema3Fdidnotpreventdifferentiationofmelanocytes,
yetmostmutantembryos(n=9/10)hadmelanocytesthatappearedto
be scattered randomly throughout the head and failed to become
properly patterned into a segmental organization with each of
thepharyngealarches(compareFig.11AandFig.11F;seeFig.S3
for mutant genotypes). Moreover, in two embryos, in addition to
the apparent random location of melanocytes, these cells were
smallwithastellateappearanceandlackedthedendriticappearance
ofmelanocytesincontrolembryos(compareFig.11BandFig.11G).
Sema3F mutant larvae also hadmaturecranial sensory ganglia,
yetthegangliainmostlarvae(n=11/12)weremalformedcompared
with controls (compare Fig. 11C,D with Fig. 11H,I) as we also
observed earlier at stage T26 (Fig. 9). These patterning defects
included misshapen ganglia (e.g. pLLG; Fig. 11I), and
Fig.8.CRISPR/Cas9knockoutofSema3Fdoesnotimpairneuralcrest unidentifiable ganglionic protrusions (? in Fig. 11I), to apparent
migration.(A,C)ControlCRISPRembryos(ContCR)showingSoxE2(A,T22) fusion of multiple ganglionic clusters [e.g. maxillomandibular
andnMyc(C,T23)expressioninmigratoryneuralcreststreams(black (mmV)+geniculate (g)+vestibuloacoustic (va), Fig. 11I]. In some
arrowheads)separatedbythin,crest-freezones(blackasterisks).(B,D)
cases, we noted individual ganglia appeared reduced in size [e.g.
Sema3FCRISPRmutantsshowingmigratoryneuralcreststreams(black
petrosal ganglion (pet), Fig. 11I], but these effects varied among
arrowheads)expressingSoxE2(B,T22)andnMyc(D,T23)segregated(black
asterisks)similartothatofcontrols.Theanteriorsideisfacingleftandthe mutantembryos.However,incontrasttodefectsincranialsensory
dorsalsideisorientedupinallpanels.Scalebars:100μm. ganglia, these embryos (n=12/12) had normally patterned trunk
neural crest-derived dorsal root ganglia and enteric neurons
embryos, including improperly patterned SoxE3+ and TwistA+ (compareFig.11EandFig.11J;seeFig.S3formutantgenotypes).
cartilage elements inpa1-2(comparepa1-2outlineinFig.10A-C Among ∼30 Sema3F mutants that survived to stage 30, larvae
withasterisksinFig.10G-I).Takentogether,theseresultshighlight developed cellular cartilage of the head and pharyngeal skeleton,
an important role for Sema3F signaling in patterning the neural including the ‘stack-of-coin’ cartilage bars in pa3-9, as well as
crest-derivedheadskeletoninlampreyembryos. mucocartilage elements in pa1-2 and the upper and lower lips
(Fig. 12). However, there were consistent and moderate to severe
patterning defects in each of these skeletal elements. In all larvae
Sema3Fsignalingisessentialforlong-termpatterningof
examined(n=15/15analyzed),themucocartilageofpa1-2failedto
pigment,cranialsensorygangliaandcartilageelementsof
properly condense into a velum, the oral skeletal element that
theheadskeleton
Finally, given that we observed early defects in the patterning of functions in agnathan respiration (compare Fig. 12A control with
cranialneuralcrest-derivedmelanocytes,cranialsensorygangliaand Sema3F mutants in Fig. 12D,G). Rather, these larvae had loosely
arranged mucocartilage and Alcian Blue-positive cellular debris
scatteredthroughouttheheadandmouth(arrowheadsinFig.12D,
G;seeFig.S3formutantgenotypes).Therewerealsoabnormalities
in the cartilage bars in pa3-9, with the most frequently observed
phenotype comprising severely bent or disjointed cartilage bars
(n=13/15)(compareFig.12B,E,H,andFig.12C,F,I).Theselarvae
also had ectopic clusters of fused cartilage nodules from adjacent
bars(arrowheadsinFig.12F,I),ordisconnectedbarsthatwerenot
fusedtogether(asterisksinFig.12F,I).
Although our gene expression analyses suggested that early
patterningoftheheadskeleton(T25-T26)wasseverelydisruptedin
Sema3FCRISPRmutants(Fig.10G-L),thelarvaeweexaminedat
T30stilldisplayedsomeevidenceofidentifiabledorsal-ventraland
Fig.9.CRISPR/Cas9knockoutofSema3Fresultsinearlymispatterning anteroposteriorpatterningofcartilagebarsinpa3-9thatallowedus
ofmelanocytesandcranialganglia.(A)T26Control(ContCR)embryo
to putatively assign some of their identities (Fig. 12E,F,H,I). We
showingnormalpatterningofmelanocyteslinearlyoverthepharynx(arrows)
attribute this difference in the severity of early- versus late-stage T
a(Bn)dSmeemlaan3oFcCytReImSPigRra(tSioenminat3oFthCeR)amntuetraionrtshehaadve(amrrioswpahteteardn)eadnmdeulpapneorclyipte(sula).t phenotypestolethalityeffectsweobservedshortlyafterT26:∼98% EN
T26,includingalackofmelanocytemigrationintotheupperlip(asterisk,B)and of5000injectedembryossurvivedtoT26(15dayspostfertilization), M
dispersedmelanocytesoverthepharynx(arrows).(C)T26controlembryo yet only ∼30 larvae (0.6%) had survived to T30 (30days post P
immunostainedforHuincranialsensoryneurons.(D)Sema3FCRISPR fertilization).Wespeculatethatthissharpincreaseinmortalitymight O
mutantsshoweddefectsinpatterningofcranialsensoryneurons,includinga beattributedtothefactthatembryoswiththemostseverelydisrupted L
lackofcondensationoftheopVganglion,apparentfusionofganglionic E
clusters(g+pet+epg?)andsplittingofinterconnectedganglia(asterisk, pharyngealdevelopmentduringearlyheadskeletonpatterningwere V
unable to properly pattern their oropharyngeal skeleton, which is E
epibranchialganglion,epg).Theanteriorsideisfacingleftandthedorsalside
D
isorientedupinallpanels.Scalebars:100μm. required for ventilation and survival at later larval stages. Thus,
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Fig.10.CRISPR/Cas9knockoutofSema3Fcausespatterningdefectsduringearlyheadskeletondevelopment.(A-F)ControlCRISPRembryos(ContCR)
atT25andT26.(A)T26embryowithSoxE1expressioninprechondrocytesofpa3-9,withweakexpressioninpresumptivemucocartilageofpa1-2(velum
outlined)andupperandlowerlips(ul,ll).(B)T26embryowithSoxE3perichondrialexpressioninthepresumptiveveluminpa1-2(velumindicatedbydashed
lines),upper/lowerlips(ul,ll),andpa3-9.(C)T25embryowithTwistAexpressioninpostmigratoryneuralcrestinpa1-2(dashedline),upperandlowerlips(ul,ll),
andpa3-9.(D-F)HorizontalsectionsthroughthepharynxofembryosinA-Cshowingpharyngealpouches(endodermoutlined),andproperlypatterned
prechondrocytes(arrowheads,D,E)orpostmigratorycrest(arrowheadsinF)surroundingthemesoderm(mesinF).(G-L)Sema3FCRISPRmutantembryos
(Sema3FCR)atT25andT26.(G)T26mutantshavedisorganizedSoxE1-postiveneuralcrestinpa3-9(arrowhead)andanabnormallyshapedoropharynx
(asterisk).(H)Sema3FT26mutantshavedisorganizedSoxE3-positiveneuralcrestinpa1-2andupper/lowerlips(asterisk)andpa3-9(arrowhead).(I)T25
mutantshaveTwistA-positivecellsscatteredinpa1-2andupper/lowerlips(asterisk),andpa3-9(arrowhead).(J-L)Horizontalsectionsthroughthepharynxof
mutantsinG-Ishowingdisorganizedprechondrocytesandpostmigratorycrestinformedorpartlyformedpharyngealarches(arrows,endodermoutlined)and
neuralcrestcellsspanningtheboundary(arrowheads)betweenpharyngealarchesinwhichtheendodermfailedtoevaginate(asterisks).Theanteriorsideis
facingleftinallpanels.DashedlinesinA-C,G-Iindicateplaneofsectionshownintheindicatedpanels.Scalebars:100μm.
larvae that survived to T30 were those that showed only moderate ganglia) within the head of lamprey embryos is driven in part by
disruptionofSema3Ffunctionandpharyngealdevelopment,which the deployment of Nrp-Sema3F signaling, an intercellular
allowedfortheirsubsequentexamination. signaling system that originated before the divergence of jawless
and jawed vertebrates over 500 million years ago. In jawed
DISCUSSION vertebrates, Sema3 proteins, especially Sema3F, function to repel
Thenewheadhypothesisproposesthattheoriginofvertebrateswas migratory and postmigratory neural crest cells that express
catalyzedbyaseriesofevolutionarymodificationstothechordatehead complementary Nrp1 and Nrp2 receptors, thereby positioning
region (Gans and Northcutt, 1983). These modifications included a groupsofneuralcresttodifferentiateintospecificderivativesalong
muscularized,pumpingpharynxthatwassupportedbyarigidcellular the embryonic anteroposterior axis (Gammill et al., 2007). Our
skeleton,aswellasaperipheralnervoussystemcontainingelaborate, results in lamprey, a basal jawless vertebrate, suggested that these
paired sensory organs (Donoghue and Keating, 2014; Gans and complementary patterns of Nrp and Sema3F expression are
Northcutt,1983).Eachoftheseinnovationswasmadepossiblebythe strikingly similar to those in jawed vertebrates and correlate with
acquisitionoftheneuralcrestand,therefore,oneoftheprimaryaimsin key stages of cranial neural crest patterning events. Moreover, our
vertebrateevolutionary-developmentalbiologyhasbeentodissectthe CRISPR/Cas9 functional experiments revealed a crucial role for
molecular,cellularandgeneticoriginsofneuralcrestcells.However,it Nrp/Sema3F-mediated patterning of multiple neural crest-derived T
N
has remained unclear how the neural crest and its underlying gene structuresinthevertebratehead,includingthecraniofacialskeleton
E
regulatory network became integrated developmentally into the and sensory ganglia, and suggest that Nrp-Sema3F signaling is a M
ancestralchordatebodyplanandacquiredtheabilitytoconstructthe deeplyconservedfunctionthatancestralvertebratesusedtopattern
P
noveltiesthatdefinethevertebrateheadandpharynx. cranial neural crest cells. In light of our findings in lamprey and O
Our gene expression and CRISPR/Cas9 functional analyses comparisons with jawed vertebrates, we propose that deployment L
E
together suggested that the ability of neural crest cells to become of intercellular guidance cues, such as Sema3, along with their V
integrated and assembled into distinct structures organized along correspondingNrpreceptors,wasinstrumentalinorganizingneural E
the anteroposterior axis (e.g. pharyngeal skeleton and cranial crest cells for the first time into derivatives in the vertebrate head D
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RESEARCHARTICLE Development(2018)145,dev164780.doi:10.1242/dev.164780
Fig.11.CRISPR/Cas9knockoutofSema3Fresultsindisorganizedmelanocytesandcranialganglia.(A-E)ControlCRISPRlarvae(ContCR)atT30.
(A)Controllarvaehavemelanocytesarrangedsegmentallyincorrelationwithpa1-9.(B)Highmagnificationofmelanocytesincontrolembryowithtypical
sizeandstellateappearance.(C)FluorescentHuimmunostainingshowingproperlypatternedcranialgangliaincontrollarva.(D)Highermagnificationimageof
insetinCshowingindividualcranialganglia.(E)FluorescentHuimmunostaininginthetrunkofcontrollarvashowingsegmentalorganizationofDRG
(arrowheads)andentericneurons(en,arrow).(F-J)Sema3Fmutantlarvae(Sema3FCR)atT30.(F)Mutantshaddisorganizedmelanocytes(arrowheads)and
(G)melanocytesweresmallerandlackedthestellatebranches(arrowheads).(H-J)Sema3Fmutantshadmispatternedcranialganglia(inset,I),butDRG
(arrowheadsinJ)andentericneurons(en,arrowinJ)appearedunaffected.QuestionmarkindicatesunidentifiableganglionicprotrusionfrompartofthemmV
ganglion.e,eye;epg,epibranchialganglion;g,geniculateganglion;mmV+g+va,fusionofgeniculateandvestibuloacousticganglia;pllg,posteriorlateralline
ganglion;va,vestibuloacousticganglion.Theanteriorsideisfacingleftandthedorsalsideisorientedupinallpanels.Scalebars:100μm.
by assuming a role in patterning events inserted temporally aredifferentiatedintoavelum,acartilaginousskeletalelementinthe
between earlier (neural crest specification) and later (neural crest agnathan oropharynx that functions in respiration (Forey, 1995;
differentiation) steps of neural crest development. Mallatt,1984,1997;Miyashita,2016;Squareetal.,2016b;Yasuiand
Once the core structural components of the neural crest (head Kaji, 2008). Our CRISPR/Cas9-mediated knockout results showed
skeletonandcranialsensoryganglia)werefixedinearlyvertebrates, thatcondensationandpatterningofthelampreyvelumrequiredproper
changesinthespatialandtemporalpatternsofintercellularsignaling Nrp/Sema3F signaling, suggesting that modifications to the early
and patterning systems via Nrp/Sema3F and others could have vertebrateoropharyngealskeletonweremediated,atleastinpart,by
enabledfurthermodificationstotheseandotherneuralcrest-derived Nrp/Sema3F signaling. In higher jawed vertebrates, the ancestrally
structures in the vertebrate head. For example, the anterior-most homonomousseriesofneuralcrest-derivedpharyngealcartilagebars
pharyngealarchesinearlyjawlessvertebratesandmodernlampreys was gradually transformed into a series of individuated structures,
T
N
Fig.12.CRISPR/Cas9knockoutofSema3Fresultsinadisorganizedheadskeleton.(A)ControlCRISPRlarva(ContCR)withAlcianBluestainingofthehead
E
skeleton.Mucocartilageelementsincludeupper/lowerlips(ul,ll),medialvelarskeleton(mvs)andpa1-2(outlined).Cartilagebarsofpa3-9areindicated. M
(B)SamelarvainAbutwithfluorescenceinpa3-9.(C)HighermagnificationimageofinsetinBshowingcartilagebarmorphology(toparrowhead)andventral
P
fusionofthebranchialbasket(bottomarrowhead).(D-F)Sema3FCRISPRmutantlarva(Sema3FCR).(D)AlcianBluestainingrevealsdisorganized O
mucocartilageofpa1-2(arrowheads).(E)Fluorescence(E,insetinF)ofthesamelarvashowsdisarticulationofcartilagebarsinpa4-6(asterisks,F)whereasthe L
middleofthesecartilagebarsareclusteredanddisjointed(arrowhead,F).(G-I)AnotherSema3FCRlarva.(G)Mucocartilagecellsarescatteredthroughoutthe E
oralskeletonthatnevercondensedintotheveluminpa1-2(arrowheads).(H)Fluorescentimaging(H,enlargedinI)ofthesamelarvashowsdisarticulated V
(asteriskinI,arch6)andfusedandbentcartilagebarsinpa3-9(arrowhead,I,arch8).Theanteriorsideisfacingleftandthedorsalsideisorientedupinallpanels. E
Scalebars:100μm. D
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RESEARCHARTICLE Development(2018)145,dev164780.doi:10.1242/dev.164780
includingjawsandhyoidaswellaselementsoftheinnerearandfacial transcription factors, such as Twist, are forcibly expressed (Abitua
skeleton(Gegenbaur,1878;Kardong,2002;Kuratani,2004;Mallatt, et al., 2012). These migratory cells colonize the pharynx as
2008;McCauleyandBronner-Fraser,2006;Romer,1950;Shigetani ectomesenchyme (Abitua et al., 2012), but there has been no gene
et al., 2005). These changes were driven primarily by spatial expression or functional analysis of the contribution of receptor-
repositioning of the ancestral pharyngeal structure and new cell- ligandguidancecuesinthesecells.Wehypothesizethatthemolecular
mesenchymeinteractions,ratherthanbytheoriginofnewcelltypes deploymentofNrp/Sema3signalingintheheadofearlyvertebrates,
(Dupretetal.,2014;Gegenbaur,1878;Gillisetal.,2013;Kardong, inconjunctionwithco-optionofotherguidanceandrepulsioncues
2002; Shigetani et al., 2002). Similarly, our functional results in into a combinatorial receptor-ligand patterning ‘code’, was an
lamprey suggest that rearrangement and novel patterning of the importantstepthatallowedstemvertebratestoorganizeneuralcrest
pharyngealandheadskeleton,alongwithothernoveltiesthroughout cellsforthefirsttimeintomanyofthehallmarktraitsthatdefinethe
vertebrateevolution(Noguchietal.,2017),wasachieved,inpart,by newvertebratehead.Therefore,itwouldbeinterestingtodetermine
alteringthespatialandtemporalactivityofacombinatorialrepulsion- whether and how migratory neural crest-like cells in invertebrate
guidance code of signaling molecules involving Sema3F, among chordatesdeployintercellularpatterningsystems.
others,forneuralcrestcellsintheheadofvertebrateembryos.
AlthoughourfindingsinlampreyimplicateanancientroleforNrp/
MATERIALSANDMETHODS
Sema3Fsignalinginpatterningneuralcrest-derivedstructuresinthe
Embryocollection
vertebrate head (cartilage, sensory neurons and pigment), we also Tocollectembryos,gravidadultsealampreys(P.marinus)wereobtained
observed importantdifferences compared with jawed vertebrates. In fromtheHammondBayBiologicalStation,Millersburg,MI,andshippedto
mouseandchickenembryos,forexample,Sema3Ffunctionsearlyin theUniversityofOklahoma.Adultswerehousedat14°Cinarecirculating
neural crest development to enforce the segregation of the three water system. Eggs were stripped manually from gravid females into a
primarycranialneuralcreststreams,whichexpressNrp1/2receptors beakerofwater(∼200ml)andmixedwithspermexpressedfromamale
(Gammilletal.,2007;Kulesaetal.,2010).Functionalperturbationof directlyontothe eggs.Embryos wererearedin smallPyrexdishesunder
constantflowindeionizedwatersupplementedwith0.05XMarc’sModified
Sema3Factivityresultsininterminglingorcompletefusionofthese
Ringers solution (MMR) chilled to 18°C.All proceduresinvolving adult
streams,leadingtoinappropriatepatterningofcranialgangliaandthe
lampreyswereperformedwithapprovalfromtheUniversityofOklahoma
pharyngealskeleton(Gammilletal.,2006,2007;Roffers-Agarwaland
InstitutionalAnimalCareandUseCommittee(IACUC,R15-027).
Gammill, 2009). By contrast, lamprey does not appear to use Nrp/
Sema3Fsignalingtopatternorsegregatemigratorycrest,anditinstead
Molecularphylogenetics
functions primarily in the structural organization of neural crest
TodeterminesequenceorthologyoflampreySema3sandNrpreceptors,we
derivatives,especiallycraniofacialcartilageandsensoryganglia.This constructedneighbor-joiningphylogenetictrees,usinggnathostomeSema7
suggestsanalternativesignalingmechanismthatmediatessegregation and Nrp and Tolloid-Like (NETO) genes as outgroups, respectively.
ofcranialneuralcreststreamsinjawlessvertebrates,althoughtheexact UntrimmedsequenceswerealignedinMEGAversion7.0usingMUSCLE,
guidance cues are unknown. Alternatively, the lack of a Sema3F and a JTT+G model for protein evolution was chosen for phylogeny
mutantphenotypeforneuralcrestmigrationmightreflecttherelaxation reconstruction (Kumar et al., 2016). Results were obtained after 1000
ofmigratoryconstraintsinthelampreyhead,aspreviouslydescribed parametric bootstrap replicates. Gene sequences analyzed and
corresponding accession numbers (in parentheses) included: Dr, Danio
(McCauleyandBronner-Fraser,2003).Inadditiontoearlypatterning
rerio (Sema3B: NP_001121818.1; Sema3C: XP_017210807.2; Sema3D:
ofmigratorycrest,Nrp/Sema3Fsignalinginjawedvertebratesisalso
AAI62510.1; Sema3Fa: AAI63764.1; Sema3Fb: AAW56082.1; Nrp1a:
crucialforpatterningtrunkneuralcrestderivatives,suchasdorsalroot
AAI63888.1; Nrp2: NP_998130.1); Gg, Gallus gallus (Sema3A:
ganglia (DRG) (Gammill et al., 2006, 2007). Similar to jawed
NP_990308.2;Sema3C:NP_989574.1;Sema3D:NP_990704.1;Sema3E:
vertebrates, lampreys also have DRG arranged along the trunk in a NP_989573.1; Sema3F: NP_989589.1; Sema3G: XP_015148335;
segmental pattern, and recent work shows that lamprey has a Sema7A: NP_001186678.1; Nrp1: NP_990113.1; Nrp2: NP_989615.1);
populationoftrunkneuralcrest-derivedentericneurons(Greenetal., Mm, Mus musculus (Sema3A: AAH90844.1; Sema3B: AAH90669.1;
2017).BothDRGandentericneuronsforminstereotypicalpositions Sema3C:NP_038685.3;Sema3D:NP_083158.3;Sema3E:NP_035478.2;
in the lamprey trunk and, therefore, are patterned using intercellular Sema3F:AAH10976.1;Sema3G:NP_001020550.1;Nrp1:AAH51447.1;
signalingcues(Greenetal.,2017).However,Nrp/Sema3Fsignaling NETO1:EDL09346.1);Pm,P.marinus(Sema3F:AAU94360.1;allother
putative lamprey Sema and Nrp sequences were obtained from manual
appears to be dispensable for patterning of the trunk neural crest
searches of the 2010 version of the sea lamprey genome assembly); Rn,
subpopulation in lamprey. This suggeststhe operation of patterning
Rattusnorvegicus(Nrp2:NP_110496.1);Xt,Xenopustropicalis(Sema3A:
cuesforneuralcrestinthelampreytrunkthataredistinctfromthosein
AAK38166.1; Nrp2: AAI36102.1); and Xl, Xenopus laevis (Sema3B:
thehead,asituationthatalsooccursinjawedvertebrates(Krulletal.,
AAI66183.1; Sema3D: NP_001087589.1; Sema3F: NP_001011157.1;
1997;Kulesaetal.,2010;Robinsonetal.,1997).Acomprehensive Nrp1:NP_001081380.1;NETO2:NP_001072912.1).
comparative analysis of awider repertoire of neural crest-patterning
mechanismsinlampreyand hagfish, anotheragnathan group, could Genecloning,insituhybridization,immunostainingandAlcian
helptoaddresswhetherancestralvertebratespatternedcranialversus Bluestaining
trunk neural crest subpopulations using distinct or overlapping PartialclonesforSema3F(553bp),NrpA1(670bp),NrpA2(550bp),NrpB
intercellularsignalingmechanisms. (584bp)andSix1/2(706bp)wereisolatedbydirectamplificationfromasea T
Althoughbonafidemigratoryneuralcrestcellsandthestructures lamprey cDNA library (primers: Sema3F forward: 5′-CCACGGAATCT- N
GGCAACCAGAA-3′; Sema3F reverse: 5′-GCGATGCGCGTGAACTT- E
thattheyformarevertebrateinnovations,thereiscompellingevidence M
GTA-3′; NrpA1 forward: 5′-CTGAGATTGTCCTGCGATTCCAC-3′,
that the closest extant relatives of vertebrates, the invertebrate NrpA1 reverse: 5′-CGCACGAACCGCGTCAGCAC-3′; NrpA2 forward: P
chordates, have ‘proto-neural crest cells’ that have a similar gene 5′-ATGCTCGCACATGTTCACAGC-3′,NrpA2reverse:5′-CGGATCAT- O
regulatory profile and can give rise to similar cell types, such as CTCTGCTGGGCG-3′;NrpBforward:5′-GGATCCTCTCGCTCTCCTT- EL
sensory neuronsand melanocytes (Abituaetal.,2012;Stolfi etal., C-3′, NrpB reverse: 5′-GGAGATGTGACAGCCGTAGA-3′; and Six1/2 V
2015).Althoughsomeofthesecellscanmigrateendogenouslyovera forward: 5′-TCCACAAGAACGAGAGCGTG-3′, Six1/2 reverse: 5′- E
shortdistance,long-rangemigrationisonlypossiblewhenneuralcrest TGCTGAGACATGTGGCTCTG-3′) (kindly provided by J. Langeland D
10