RESEARCH ARTICLE

The Formation of the Superior and JugularGanglia: Insights Into the Generation ofSensory Neurons by the Neural CrestHannah Thompson,1† Aida Blentic,1 Sheona Watson,1‡ Jo Begbie,2 and Anthony Graham1*

The superior and jugular ganglia (S/JG) are the proximal ganglia of the IXth and Xth cranial nerves andthe sensory neurons of these ganglia are neural crest derived. However, it has been unclear the extent towhich their differentiation resembles that of the Dorsal Root Ganglia (DRGs). In the DRGs, neural crestcells undergo neuronal differentiation just after the onset of migration and there is evidence suggestingthat these cells are pre-specified towards a sensory fate. We have analysed sensory neuronal differentia-tion in the S/JG. We show, in keeping with previous studies, that neuronal differentiation initiates long af-ter the cessation of neural crest migration. We also find no evidence for the existence of migratory neuralcrest cells pre-specified towards a sensory phenotype prior to ganglion formation. Rather our resultssuggest that sensory neuronal differentiation in the S/JG is the result of localised spatiotemporal cues.Developmental Dynamics 239:439–445, 2010. VC 2009 Wiley-Liss, Inc.

Key words: superior and jugular ganglia; neural crest cells; neuronal differentiation

Accepted 31 October 2009

INTRODUCTION

The route through which neural crest

cells give rise to sensory neurons has

been most extensively analysed in the

trunk, where it has been shown that

there is a correlation between the tim-

ing and pathway of migration and fate

(Le Douarin and Kalcheim, 1999).

Thus, the neural crest cells that form

the sensory ganglia are those that

migrate early and move ventrally into

the anterior half sclerotome of each

somite as a result of being repulsed by

the posterior sclerotome. These neural

crest cells subsequently differentiate

within the anterior sclerotome to form

sensory neurons and glia. By contrast,

those that form autonomic ganglia

migrate further ventrally and stop in

the vicinity of the dorsal aorta, while

those that form melanocytes migrate

last and move dorsolaterally between

the dermamyotome and the ectoderm.

Although there is a clear correlation

betweenmigration and fate in the neu-

ral crest, there has long been a debate

as to how the sensory lineage is estab-

lished, particularly with respect to the

time at which neural crest cells are

specified to become sensory neurons.

A number of older studies support

the view that migratory neural crest

cells are multipotent and that they

only become restricted to a sensory

fate after migrating into the anterior

somitic sclerotome as a result of envi-

ronmental cues. In vitro studies have

demonstrated the existence of individ-

ual neural crest cells that are capable

of giving rise to sensory neurons and

other derivatives (Le Douarin et al.,

2004). Furthermore, in vivo cell label-

ling experiments have demonstrated

the existence of neural crest cells that

will give rise to both sensory and

1MRC Centre for Developmental Neurobiology, King’s College London, London, United Kingdom2Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom†H. Thompson’s present address is Department of Veterinary Basic Sciences, Royal Veterinary College, London, UK.‡S. Watson’s present address is Division of Immune Cell Biology, National Institute for Medical Research, London, UK.Grant sponsors: MRC (UK), Wellcome Trust.*Correspondence to: Anthony Graham, MRC Centre for Developmental Neurobiology, King’s College London, London,United Kingdom. E-mail: anthony.graham@kcl.ac.uk

DOI 10.1002/dvdy.22179Published online 11 December 2009 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 239:439–445, 2010

VC 2009 Wiley-Liss, Inc.

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autonomic neurons (Fraser and Bron-

ner-Fraser, 1991). It has also been

suggested that the neural tube may

play a prominent role in driving sen-

sory neurogenesis (Kalcheim and Le

Douarin, 1986). If a silastic barrier

was inserted between the neural tube

and the recently migrated neural

crest cells, then the Dorsal Root Gan-

glion (DRG) fails to form. This opera-

tion, however, had no effect on the for-

mation of sympathetic neurons or

more ventrally located Schwann cells.By contrast, there is also an accu-

mulating body of work suggestingthat neural crest cells fated to becomesensory neurons are restricted intheir potential at early stages. A sub-set of neural crest cells apparently re-stricted to a sensory neuronal fatehas been described. It has been sug-gested that the basic helix-looop-helixtranscription factors, the Neurogenins(Ngn1 and Ngn2), are involved in theearly specification of the sensory line-age. Ngn2 is expressed by migratingneural crest cells early in their migra-tion and cre-recombinase fate map-ping of these cells suggests that theyare biased to follow a sensory fate(Gradwohl et al., 1996; Sommer et al.,1996; Zirlinger et al., 2002). More-over, in single Ngn2 and doubleNgn2;Ngn1 mutants sensory neuro-genesis fails, but sympathetic gangliaare unaffected (Ma et al., 1999). It hasalso been proposed that Gdf7, a Bmp

Fig. 1. The cranial sensory gangla. Side viewof an isolated hindbrain with associated sen-sory ganglia immunostained with HuC/D atstage 28 highlighting the positions of the cra-nial sensory ganglia. The superior (S) and thejugular (J) ganglia are located at the proximalend of the IXth and Xth nerves, respectively.The placodally derived ganglia are also appa-rent: the trigeminal (T), the vestibuloacoustic(VA), the geniculate (G), the petrosal (P), andthe nodose (N).

Fig. 2.

Fig. 3.

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family member exclusively expressedin the dorsal aspect of the neuraltube, acts to restrict neural crest cellsto a sensory fate as they emigratefrom the neural tube. Lineage tracingsuggests that Gdf7-expressing premi-gratory neural crest cells will follow asensory fate and that this ligand canpromote sensory neuronal differentia-tion in cultures of neural crest cells(Lo et al., 2005).

It is, however, unclear if the lessonslearned from the trunk are generallyapplicable to the generation of all sen-sory neurons by neural crest cells. Toaddress this issue, we have focussedon the development of the superiorand jugular ganglia. These are theproximal ganglia of the IXth and Xthcranial nerves, respectively (Fig. 1).The sensory neurons of these gangliaare neural crest derived (D’Amico-Martel and Noden, 1983), and theyfunction to relay information from theairways and blood vessels.

Importantly, the development of thesuperior and jugular ganglia differs ina number of significant ways fromthose of the DRG. These ganglia formin a somite-free environment. Theneurons of these ganglia differentiatejust caudal of the otic vesicle but ros-tral to the somites, at the site of entry/exit for the IXth and Xth cranialnerves, the glossopharyngeal and

vagus, respectively. Furthermore, incontrast to what is seen in the trunk,the neural crest cells that generatethe sensory neurons of these gangliaare the late migrating population(Baker et al., 1997). At this axial level,the early migrating neural crest cellsfill the pharyngeal arches and gener-ate ectomesenchymal derivatives.Finally, sensory neuronal differentia-tion is believed to occur some daysafter the cessation of neural crestmigration (D’Amico-Martel, 1982).This contrasts with the situation seenduring the formation of the DRGswhere sensory neuronal differentia-tion follows on from the migration ofthe neural crest cells into the anteriorsclerotome of the somites (Marusichet al., 1994). Thus, with respect to thedifferentiation of the neurons of thesuperior and jugular ganglia, two al-ternative scenarios could be envis-aged. It may be that neural crest cellsare pre-specified as they migrate fromthe neural tube but that, in contrast tothe situation seen in the DRG, differ-entiation is stalled for a prolonged pe-riod. Alternatively, the neural crestcells that form the neurons of the supe-rior and jugular ganglia may not bepre-specified towards a sensory fate asthey migrate but are induced toundergo sensory neuronal differentia-tion by local signals that act at rela-

tively late stages. In this study, we testthe validity of these scenarios and wepresent evidence that supports thelatter.

RESULTS

Timing of the Onset of

Neuronal Differentiation in

the Superior and Jugular

Ganglia

The superior and jugular ganglia (S/JG) are the neural crest–derived prox-imal sensory ganglia of cranial nervesIX and X that form just caudal of theotic vesicle in the vicinity of the entry/exit point of these cranial nerves (Fig.1). Previous studies, using 3H-thymi-dine incorporation, have shown thatneuronal differentiation in the S/JGstarts at around day 4 of chick embry-onic development (D’Amico-Martel,1982). To more precisely map the dif-ferentiation of these neurons, we usedan antibody that labels neurons fromtheir birth date onwards, HuC/D, aswell as analysing the expression ofISL1 a transcription factor that iswidely expressed by peripheral nerv-ous system neurons (Marusich et al.,1994). Our previous studies haveshown that both HuC/D and ISL1expression in the cranial ganglia arefirst apparent in the placodally-derived neurons starting at stage 14(Hunter et al., 2001; Begbie et al.,2002, 2004). Subsequently, expressionis then established in the formingDRGs of the trunk. Thus, by stage 21,HuC/D and Isl1 expression can bedetected in the Trigeminal, Vestibu-loaccoustic, Geniculate, Petrosal, andNodose ganglia of the head and in theDRGs at cervical, thoracic, and lumberlevels (Fig. 2A, B). At this stage, thereis no expression at the site of forma-tion of the S/JG . However, HuC/D-and ISL1-positive neurons can first beobserved at this position from stage 22(Fig. 2C–F). These neurons are locatedat the site of the forming superior gan-glion, just caudal of the otic vesiclelying directly alongside the neuroepi-thelium. As development progresses,the number of neurons in the formingS/JG increases and the gangliabecomes readily apparent (Fig. 1).Thus, we have pinpointed the initia-tion of neuronal differentiation in theS/JG at stage 22, towards the end of

Fig. 2. The differentiation of the neurons of the Superior and Jugular ganglia. A: Whole-mountHuC/D staining of a stage-21 embryo highlighting the neurons of the trigeminal (T), vestibuloa-coustic (VA), geniculate (G), petrosal (P), and nodose (N) ganglia, and the DRGs of the trunk.B: Whole-mount in situ hybridisation with ISL1 highlighting the neurons of the trigeminal (T), ves-tibuloacoustic (VA), geniculate (G), petrosal (P), and nodose (N) ganglia, and the DRGs of thetrunk. In A and B, the position of the DRGs lying at the level of the forelimb is indicated by whitearrowheads. C: Transverse confocal section through the post-otic hindbrain (HB) of a stage-22embryo showing the accumulation of HuC/D expressing post-mitotic neurons of the formingsuperior ganglion alongside the neural tube. D: Magnification of area boxed in C. Arrowheadpoints to emerging neurons of the superior ganglion. E: Transverse confocal section through thepost-otic hindbrain (HB) of a stage-22 embryo showing the accumulation of ISL-1-expressingneurons of the forming superior ganglion alongside the neural tube. F: Magnification of areaboxed in E. Arrowhead points to emerging neurons of the superior ganglion. The sensory neuro-nal transcription factors BRN3A (G) and DRGX (H) are expressed in the superior ganglia (whitearrowheads) at stage 26.

Fig. 3. Expression of early markers of sensory neuronal differentiation. SOX10 (A, B) andFOXD3 (C, D) in situ hybridisation showing that there are many neural crest cells in the post-oticregions at the entry/exit points of the IXth and Xth cranial nerves at stage 18 (A, C) and thatsome of these are abutting the hindbrain (B, D). At stage 16, there is pronounced expression ofGDF7, a factor implicated in the induction of sensory neurons, in the dorsal region of the hind-brain (E, F). There is, however, no expression of either NGN1 (G) or NGN2 (H), both of which areinvolved in the early specification of the sensory lineage, in cells in the vicinity of the post-otichindbrain (arrowhead). NGN1 is, however, expressed in the maxillomandibular trigeminal (Tm)placode and vestibuloacoustic region of the otic vesicle (G). NGN1 is also expressed by neuronswithin the caudal hindbrain (H). NGN2 is expressed in the ophthalmic trigeminal placode (I). OV,otic vesicle; HB, hindbrain.

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the third day of development andslightly earlier than previouslydescribed. Furthermore, our studiesconfirm the observation that differen-tiation of these sensory neurons occurssubstantially after neural crest emi-gration from the caudal hindbrain,which takes place from stage 10 of de-velopment, and that it lags signifi-cantly behind DRG neuronal differen-tiation (Ferguson andGraham, 2004).

HuC/D and ISL1 are generic neuro-nal markers; therefore, to comparesensory neurogenesis in the S/JGwith DRG development we have alsoused 2 markers specific to sensoryneuron development found in the ma-jority of DRG neurons: BRN3A andDRGX (Fedtsova and Turner, 1995;Saito et al., 1995).

Our expression analysis shows thatboth BRN3A (Fig. 2G) and DRGX(Fig. 2H) are expressed in the cells ofthe forming superior and jugular gan-glia. These results confirm that theneurons of the S/JG express a similarrepertoire of transcription factors tothose of the DRG.

Expression of Early

Indicators of Sensory

Neuronal Differentiation

A possible reason for the delay in neu-ronal differentiation is that neuralcrest cells are not positioned at thesite of ganglion formation till rela-tively late stages. We have closelyexamined the distribution of the neu-ral crest cells via expression of SOX10and FOXD3, two key transcriptionfactors expressed by early neuralcrest cells (Britsch et al., 2001). Herewe show that at stage 18 there aremany neural crest cells, expressingSOX10 and FOXD3, lying alongsidethe caudal hindbrain at the sites ofentry/exit for the IXth and Xth cra-nial nerves, the sites of formation ofthe superior and jugular ganglia, aswell as associating with the formingnerves (Fig. 3A–D).

A further explanation for the delayin sensory neuronal differentiationcould be the absence of the signallingmolecules involved in directing sen-sory neuronal differentiation. Theidentification of signalling moleculesthat direct sensory neuronal fate has

made some progress in recent years.WNTs have been found to have a rolein directing neural crest cells to adopta sensory fate (Lee et al., 2004). How-ever, numerous studies have shownthat WNTs are expressed in the dor-sal neural tube all along the neuraxisfrom early stages (Wu et al., 2003),and so their expression long precedessuperior and jugular sensory neuro-nal differentiation. Studies in micehave also suggested that Gdf7, whichis expressed in the roof plate, biasesneural crest cells towards a sensoryfate and can promote sensory neuro-nal differentiation of neural crest cellsin vitro (Zirlinger et al., 2002). Theexpression of this gene has not beenanalysed in the roof plate of the hind-brain with respect to the formation ofthe superior and jugular ganglia. We,therefore, detailed its expression pat-tern. We find that GDF7 expression islocalised to the roof plate of the hind-brain from early stages, and it isapparent from transverse section thatthe expression of this gene is re-stricted to the point at which the roofplate abuts the dorsal aspect of thehindbrain (Fig. 3E, F). Thus, GDF7expression, like that of the WNTs, isestablished in the roof plate well inadvance of superior and jugular neu-ronal differentiation.

It has also been suggested that theneurogenins, NGN1 and NGN2, areinvolved in the early specification ofthe sensory lineage. Given that sig-nalling molecules involved in promot-ing sensory neuronal differentiationare expressed early, it is possible thatalthough terminal differentiation hasnot occurred, the neural crest cellshave initiated the sensory differentia-tion programme. To assess this possi-bility, we analysed the expression ofNGN1 and NGN2. We find thatalthough these genes are expressed atstage14 in the neurogenic placodes,there is no expression in the neuralcrest cells flanking the caudal hind-brain (Fig. 3G–I), although there isexpression of NGN1 within the hind-brain (Fig. 3H). Thus, they exhibit noindication of having already initiatedsensory neuronal differentiation. Thisresult is in keeping with the observa-tion in mouse that Ngn1 expression isfirst detected in the S/JG at E10.5,which is also long after the cessation

of neural crest migration in this spe-cies (Ma et al., 1998).

Timing of Entry/Exit of

Cranial Nerves IX and X

Relative to Sensory Neuron

Differentiation in the

Superior and Jugular

Ganglia

Our data suggest that the neuralcrest progenitors of the S/JG are inplace for a prolonged period beforethe sensory neurons differentiate. Tobegin to address what may triggerneuronal differentiation in these neu-ral crest cells, we have examinedtheir environment to identify poten-tial cues. The positioning of the neu-ral crest cells at the entry/exit pointof nerves IX and X suggests that onesuch cue may be the establishment ofthe projections of the motor and sen-sory neurons that contribute to thesenerves. Indeed, studies on the forma-tion of the trigeminal ganglion havesuggested that the normal differentia-tion of the placodally-derived neuronsis important for the differentiation ofthe neural crest–derived neurons(Hamburger, 1961; Moody and Hea-ton, 1983). We have, therefore, usedneurofilament middle chain (NF-M)immunostaining to determine thetiming of exit of motor projections andentry of placodal sensory projectionswith respect to neuronal differentia-tion in the S/JG.At stage 16, NF-M staining high-

lights the forming placodally-derivedsensory ganglia: trigeminal, vestibu-loacoustic, geniculate, and petrosal,as well as many CNS-derived neurons(Fig. 4A–C). It is further apparent, insection, that glossopharyngeal (IXth)efferent fibres are exiting the neuraltube at this stage (Fig. 4C). The affer-ent fibres of the IXth nerves, whichare supplied by cells in the petrosalganglion, have not as yet penetratedthe brainstem (Fig. 4C). By stage 18,however, there are many axons run-ning into the neural tube from petro-sal neurons and thus the entry/exitpoint of the IXth nerve is well estab-lished (Fig. 4D–F). At this stage, theentry/exit point of the Xth nerve hasas yet to form, but is formed by stage20 (data not shown). Thus, the entry/

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exit points of the IXth and Xth cranialnerves are formed well in advance ofthe differentiation of the neurons ofthe superior and jugular ganglia.

DISCUSSION

In this study, we have scrutinised thedifferentiation of the sensory neuronsof the superior and jugular ganglia,and in doing so we have contrastedthis process with DRG sensory neuro-nal differentiation. Intriguingly, wefind a number of important differen-ces between the superior and the jug-ular ganglia and the DRGs. As notedbefore (D’Amico-Martel, 1982), wefurther detail the fact that neuronaldifferentiation initiates at a relativelylate time with respect to neural crestemigration. Thus, while neuronal dif-ferentiation follows on from neuralcrest emigration in the DRG, theseprocesses are separated by about 2days in the superior and jugular gan-glia. Yet, we also show that neuralcrest cells have populated the sites atwhich the superior and jugular gan-glia will form. Furthermore, thesecells do not express any of the genesassociated with sensory neurogenesis

until stage 22 of development, eventhough signalling molecules impli-cated in DRG sensory neurogenesisare present from early stages. Thus,our results strongly support the viewthat sensory neuronal differentiationin the superior and jugular gangliais not the result of the pre-specifica-tion of migratory neural crest cellstowards a sensory phenotype butrather may be triggered, at aroundstage 22 of development, by a local-ised signal.

There have been two studiesemploying cre-based fate mappingthat suggest the existence of a subpo-pulation of neural crest cells biasedtowards a sensory neuronal fate. Thefirst analysedNgn2-expressing neuralcrest cells and the second neural crestcells derived from roof plate cellsexpressing Gdf7. Our studies, how-ever, find no indication of the existenceof such a population in the post-oticcrest. We find at this axial level thatneurogenin expression is restricted toplacodal cells and CNS neurons atearly stages. Furthermore, we findthat GDF7 is expressed in the dorsalneural tube during the period ofneural crest emigration, yet sensory

neuronal differentiation does not com-mence till much later. These observa-tions suggest that the control of sen-sory neuronal differentiation in thepost-otic neural crest is very distinctfrom that in the trunk.Rather than finding support for the

view that neural crest cells are pre-specified to become sensory cells, ourstudies clearly support the view thatsensory neuronal differentiation isdriven by environmental cues. Such aview has previously been espoused fortrunk neural crest cells with the neu-ral tube being identified as the sourceof the signalling for triggering sen-sory differentiation (Kalcheim and LeDouarin, 1986). Our results wouldstrongly suggest that such a scenariois likely to apply to the superior andjugular ganglia. We find no indicationof sensory neuronal differentiationoccurring till a specific late timepoint. We also find that the first neu-rons to differentiate do so immedi-ately adjacent to the neural tube.It has also been argued, however,

that the placodally derived sensoryneurons are key for the differentiationof the neural crest–derived sensoryneurons (Hamburger, 1961) and as

Fig. 4. Timing of entry/exit of the afferent and efferent fibres of the IXth and Xth cranial nerves. We have used NF-M immunostaining to detail thedevelopment of the IXth and Xth cranial nerves. At stage 16 of development, the formation of the trigeminal, vestibuloacoustic, and geniculateganglia is well underway. However, the petrosal ganglion is just forming (A, B). A transverse section reveals that while motor fibres of the IXthnerve are exiting the neural tube, afferents from petrosal neurons have failed to penetrate at this stage (C) . By stage 18, the cranial nerve devel-opment has advanced (D) and at this stage afferent projections from the petrosal ganglion are entering the hindbrain (E, F). T, trigeminal; VA, vesti-buloacoustic; G, geniculate; P, petrosal; N, nodose.

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such the signal that triggers superiorand jugular neuronal differentiationcould be axonal projections from theneurons of the distal, placodally-derived ganglia of the IXth and Xthnerves, the petrosal and nodose. How-ever, we find these afferent fibres arein place well in advance of superiorand jugular differentiation. It is alsoimportant to appreciate that the con-clusion that placodally derived neu-rons are important for the differentia-tion of neural crest–derived sensoryneurons was based on studies of thetrigeminal ganglion where these twopopulations are juxtaposed, and exper-imental manipulation of one popula-tion is likely to impact upon the other.This situation does not apply to theganglia of the IXth and Xth cranialnerves, as the placodally derived sen-sory neurons are quite distant fromthe neural crest–derived ganglia.

Our observations are very much inkeeping with the extensive body ofevidence demonstrating plasticity inthe cranial neural crest (Le Douarinet al., 2004). It would seem unlikelythat sensory neuronal differentiationin the head is driven via prespecifica-tion of neural crest cells towardsthat fate. Rather, environmental cuesprobably play a pre-eminent role.Thus, there would seem to be sub-stantive differences between the pro-cess of sensory neuronal differentia-tion in the DRGs versus the superiorand jugular ganglia.

EXPERIMENTAL

PROCEDURES

Whole-Mount In Situ

Hybridisation and

Immunohistochemistry

Fertile hens eggs were incubated in ahumidified chamber at 37�C andstaged according to Hamburger andHamilton (1992). Extra-embryonicmembranes were removed before fix-ing in 4% PFA in PBS overnight. Forwhole-mount in situ hybridisation,embryos were washed twice in PBST(PBS þ 0.1% Tween-20), then dehy-drated through 25 to 100% methanolin PBST, bleached with 6% H2O2 inmethanol for 1 hr. They were thenrehydrated in 75–25% methanol andtreated with 10 mg/ml Proteinase K in

PBST for 20 min, postfixed with 4%PFA/0.1% gluteraldehyde for 20 min,and then washed twice in PBST. Theembryos were incubated at 70�C inhybridisation buffer (50% formamide,1.3� SSC, 5 mM EDTA, 50 mg/mltRNA, 100 mg/ml heparin, 0.2%Tween-20, 0.5% CHAPS) for 1 hr,followed by overnight at 70�C in di-goxigenin-labelled riboprobes, sythes-ised following the manufacturer’sinstructions (Roche, Nutley, NJ)against ISL1, BRN3a, DRGX,SOX10, FOXD3, GDFf7, NGN1, orNGN2, diluted 10 ml in 1 ml wholemount–hybridisation buffer. Embryoswere washed four times with wholemount–hybridisation buffer at 70�C,then three times 30 min with MABT(100 mM maleic acid, 150 mM NaCl,1% Tween-20; pH 7.5), and blockedwith 2% BBR (Roche)/20% goat serum/MABT and incubated overnight inanti-DIG-AP antibody (Roche) diluted1:2,000 in the same block. Embryoswere washed extensively with MABTand the alkaline phosphatase activitydetected using NBT and BCIP inNTMT. The reaction was stopped bywashing in MABT and fixing in 4%PFA. Immunostaining was done usingstandard whole-mount immunostain-ing protocol (Mackenzie et al., 1998).Embryos were washed three timesin PBSTx (PBSþ1% TritonX-100)and blocked with 10% goat serum/PBSTx for 90 min, followed by a 1-week incubation in primary antibody(mouse anti-HUC/D (MolecularProbes, Eugene, OR; 1:300), mouseanti-Isl1/2 (DSHB, Iowa City, IA;1:1,000), and mouse anti-neurofila-ment middle chain (Zymed, San Fran-cisco, CA; 1:10,000) in the same block-ing solution. Embryos were washedthree times in block and then incu-bated for 2 days in an appropriateAlexa secondary antibody (MolecularProbes; 1:1,000). Embryos were thenwashed in PBSTx, bisected or sec-tioned, mounted using 90% glycerolPBS, and imaged using a compound orconfocal microscope. For sectioning,chick embryonic heads were placed in20% gelatine/PBS and left to infiltratefor 1 hr at 55�C, embedded at roomtemperature, and left on ice for 30 minbefore fixing overnight in ice-cold 4%PFA/ PBS. Sections were cut in trans-verse at 50 mm on a vibrotome and

mounted on slides (VWR) using 90%glycerol PBS.

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