Research Article Development of the Early Axon Scaffold in ...downloads.hindawi.com/archive/2014/196594.pdf · Research Article Development of the Early Axon Scaffold in the Rostral

Research ArticleDevelopment of the Early Axon Scaffold in the Rostral Brain ofthe Small Spotted Cat Shark (Scyliorhinus canicula) Embryo

Michelle Ware12 Colin P Waring3 and Frank R Schubert1

1 Institute of Biomedical and Biomolecular Science University of Portsmouth PO1 2DY Portsmouth UK2 Institut de Genetique et Developpement CNRS UMR6290 Faculte de Medecine Universite de Rennes 1 35000 Rennes France3 Institute of Marine Sciences School of Biological Sciences University of Portsmouth P04 9LY Portsmouth UK

Correspondence should be addressed to Michelle Ware michellewareuniv-rennes1fr

Received 2 July 2014 Revised 15 September 2014 Accepted 16 September 2014 Published 29 October 2014

Academic Editor Guillermo Estivill Torrus

Copyright copy 2014 Michelle Ware et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The cat shark is increasingly used as a model for Chondrichthyes an evolutionarily important sister group of the bony vertebratesthat include teleosts and tetrapods In the bony vertebrates the first axon tracts form a highly conserved early axon scaffold Thecorresponding structure has not been well characterised in cat shark and will prove a useful model for comparative studies Usingpan-neuralmarkers the early axon scaffold of the cat shark Scyliorhinus canicula was analysed Like in other vertebrates themediallongitudinal fascicle was the first axon tract to form from a small cluster of neurones in the ventral brain Subsequently additionalneuronal clusters and axon tracts emerged which formed an array of longitudinal transversal and commissural axons tracts inthe Scyliorhinus canicula embryonic brain The first structures to appear after the medial longitudinal fascicle were the tract of thepostoptic commissure the dorsoventral diencephalic tract and the descending tract of themesencephalic nucleus of the trigeminalnerve These results confirm that the early axon scaffold in the embryonic brain is highly conserved through vertebrate evolution

1 Introduction

The initial nerve connections that develop during earlydevelopment of the rostral brain form a structure that hasbeen termed the early axon scaffold [1] This structure hasremained highly conserved through the vertebrate taxaThese tracts act as pioneers for the later follower axons andallow more complex connections to be made The early axonscaffold has been well described in non-jawed vertebratessuch as lamprey [2] and jawed bony vertebrates such aszebrafish Xenopus chick and mouse [1 3ndash5] Briefly theearly axon scaffold forms from small clusters of neuronesin distinct regions of the brain which project axons forminglongitudinal transversal and commissural tracts In all ver-tebrates studied apart from mouse the medial longitudinalfascicle (MLF) forms first from a cluster of neurones in thebasal diencephalon In mouse the descending tract of themesencephalic nucleus of the trigeminal nerve (DTmesV)neurones appears first closely followed by the appearanceof MLF neurones [4 6] In anamniotes the prosencephalon

contains an array of tracts and commissures such as theanterior commissure (AC) supraoptic commissure (SOT)postoptic commissure (POC) and tract of the postopticcommissure (TPOC)

Analysis of cartilaginous fish is particularly interestingas this is the sister group to bony vertebrates Similaritiesbetween the two branches of gnathostomes provide an insightinto the last common ancestor of jawed vertebrates For thisreason the group is often utilised in origin and evolutionstudies by comparative embryologists In this study the smallspotted cat shark (or lesser spotted dog shark) an emergingmodel for studying and comparing brain development hasbeen used While the adult brain connections have been welldescribed in cartilaginous fish a description of the initialaxon tracts is lacking [7] Neuronal development of specificneurone types has been studied in this cat shark but at laterstages after the early axon scaffold has formed [8ndash12] Theearly axon scaffold has been mentioned in the cat sharkspecies Scyliorhinus torazame but this study only showed

Hindawi Publishing CorporationInternational Scholarly Research NoticesVolume 2014 Article ID 196594 8 pageshttpdxdoiorg1011552014196594

2 International Scholarly Research Notices

the formation at an older stage (Stage V) once the early axonscaffold appeared to be fully established [13]

The aim of this study was to characterise the early axonscaffold through a time series in the cat shark speciesScyliorhinus canicula using immunostaining

2 Materials and Methods

21 S canicula Embryos Sexually mature cat sharks (Scanicula) were caught locally in the summer off the coastof Portsmouth Hampshire UK and kept in a flow-through500 L seawater tank under ambient conditions at the Insti-tute of Marine Sciences University of Portsmouth Femalesregularly laid eggs wrapped around the outflow pipes of thetank S canicula eggs were incubated in flow-through sea-water at approximately 17∘C until they reached the requiredembryonic stage [14] To harvest the embryo a window wascut into the top egg to allow access The embryos werewashed in phosphate buffered saline (PBS) and fixed with4 paraformaldehydePBS or MEMFA (01M MOPS2mMEGTA1mM MgSO

437 formaldehyde 30ndash40 minutes

washed and stored in methanol)

22 Immunohistochemistry S canicula embryos were pre-pared for immunofluorescence by opening the rhomben-cephalon and the telencephalic vesicles The axon tractswere visualised using pan-neural antibodies Tuj1 mouse(Abcam ab7751 1 1000) or rabbit (Abcam ab18207 1 1000)and the neuronal populations specifically with anti-HuCD(Molecular Probes A21271 1 500) Primary antibodies werevisualised with Alexa fluorochrome-conjugated IgG anti-mouse (Invitrogen A11001 1 500) or anti-rabbit antibody(Invitrogen A11008 1 500) The protocol for immunohisto-chemistry has been described previously [15]

23 DiO Labeling Cyanine dye DiO (331015840-dioctade-cyloxacarbocyanine perchlorate Molecular Probes D275)was used to trace theMLF axons from the rhombencephalonS canicula embryonic brains were injected with dye aspreviously described [5]

24 Microscopy and Image Processing A Zeiss Stereo LumarV12 fluorescent stereomicroscope was used to obtain lowmagnification images of the embryos For more detailedimages Zeiss LSM 510 and LSM 710 confocal microscopeswere used Images were processed using Axiovision Rel 46ImageJ and Photoshop CS software

3 Results

S canicula embryos have been studied between stages 18 and25 [14] In terms of early axon development these stages wereequivalent to zebrafish stages 16 hours post fertilisation (hpf)to 24 hpfXenopus stages 22 to 32 chick stages HH11 toHH18and mouse stages E85 to E105 (data not published)

31 Detailed Description of the First Neurones to Differentiatein the Embryonic S canicula Brain Using an antibody against

120573III tubulin (Tuj1) the first neurones were detected withinthe S canicula embryonic brain at stage 18 (Figure 1(a) darrow) These neurones will form the nucleus of the MLF(nMLF) and occupied a location similar to theMLF neuronesin the chick brain [5] presumably rostral to the diencephalic-mesencephalic boundary (DMB) The MLF neurones startedprojecting axons caudally at stage 19 (Figure 1(b) e)TheMLFcontinued to project axons caudally at stage 20 (Figure 1(f))Also evident at stage 20 (Figure 1(f) unfilled arrow) and stage21 (Figure 1(g) unfilled arrow) were a scattered populationof neurones located more rostrally compared with the initialMLF neurones but still projecting axons into the MLFtract By stage 21 the MLF had formed a tight bundle thatprojected along the floor plate into the rhombencephalon(Figure 1(g) and data not shown) To confirm that the MLFhad reached the rhombencephalon the MLF axon tract waslabelled with DiO from the rhombencephalon at stage 22(Figure 1(c) orange spot) The dye diffused back to neuroneslocated in the diencephalon that belonged to the nMLF(Figure 1(l)) This confirmed that the axons of the MLFhad reached the rhombencephalon by stage 22 At stage 23the MLF was well established and appeared to be formedfrom three populations of neurones (Figures 1(h) 1(j) and1(k)) Double labelling with Tuj1 and HuCD revealed onepopulation thatwas located along the floor plate (Figures 1(h)1(j) and 1(k) arrow) another population that was locatedmore dorsally (Figures 1(j) and 1(k) unfilled arrowhead) andthe rostrally located scattered neurones (Figure 1(h) unfilledarrow)The population of neurones along the floor plate weretightly clustered and appeared to be contributing most ofthe axons to the MLF while the dorsal population was morescattered like the more rostral population There were axonsprojecting dorsally to the MLF axon tract and appeared to bea separate tract (Figure 2(j) arrowhead) These axons mostlikely projected from neurones that were part of the rostralMLF scattered population By stage 25 the MLF was wellestablished and the ventral commissure (VC) had formedacross the ventral midline (Figure 1(i))

32 Formation of the DTmesV DVDT and TPOC in theEmbryonic S canicula Brain Interestingly until stage 23 theMLF was still the only prominent tract in the S caniculabrain (Figures 1(a)ndash1(c)) Although it was not clear fromthe overview image at stage 23 (Figure 2(a)) higher mag-nification with double labelling of Tuj1 and HuCD anti-bodies revealed that there were other axon tracts beginningto form (Figures 2(b)ndash2(d)) Neurones appeared along thedorsal midline of the alar plate in the mesencephalon thatformed the nucleus of the DTmesV (nmesV) giving rise tothe DTmesV (Figure 2(b) arrow) There were 3-4 neuroneslocated dorsally at the epiphysis (Figure 2(c) arrow) Theseneurones started projecting axons ventrally pioneering thedorsoventral diencephalic tract (DVDT) One of these neu-rones had projected an axon ventrally almost reaching theMLF (Figure 2(c) arrowhead) The tract of the postopticcommissure (TPOC) formed from neurones located in therostral hypothalamus the nucleus of the TPOC (nTPOC)(Figure 3(d)) There were also neurones located in the dorsal

International Scholarly Research Notices 3

Stage 18

prosmes

dos

(a)

pros mese

Stage 19

nMLF

rh

(b)

pros mes

op

Stage 22

MLFrh

(c)

Stage 18

(d)

Stage 19

MLF

(e)

Stage 20

MLF

(f)

Stage 21

MLF

Tuj1

(g)

Stage 23

MLF

(h)

Stage 25

MLFVC

(i)

Stage 23

MLF

Tuj1

HuC

D

(j)

Stage 23

nMLF

(k)

Stage 22

MLF

DiO

(l)

Figure 1 Detailed formation of theMLF in the embryonic S canicula brain Lateral view of wholemount embryonic brain Scale bars 100 120583m(a)ndash(c) Overview of embryos (a) Stage 18 The first neurones appeared at the ventral DMB (arrow) These neurones formed the nMLF Boxindicates high magnification in D (b) Stage 19 The number of MLF neurones has increased Box indicates high magnification in E (c) Stage22 The MLF was still the prominent tract with scattered neurones located rostrally projecting axons into the MLF (unfilled arrow) Orangespot indicated whereDiOwas injected (d) Stage 18The firstMLF neurones appeared (arrow) (e) Stage 19TheMLF neurones were projectingtheir first axons caudally (f) Stage 20 TheMLF axons have projected further caudally and the first rostrally located scattered MLF neuronesappeared (unfilled arrow) (g) Stage 21TheMLF axons have formed a tight bundleThe scatteredMLF neurones were projecting into theMLFtract (unfilled arrow) (h) Stage 23TheMLF has increased in number of axons and neurones ScatteredMLF neurones projected into theMLFaxon tract (unfilled arrow) A tight population of MLF neurones was present ventrally (arrow) (i) Stage 25 The MLF was well establishedand the VC was also present (j) Stage 23 double labelling with Tuj1 and HuCD Caudal to the scattered MLF neurones the MLF nucleusappeared to be arranged into a further two populations of MLF neurones one located dorsally (unfilled arrowhead) and the other as a tightcluster of centrally located neurones along the ventral midline (arrow) There were also axons projecting ventrally to the MLF (arrowhead)that appeared to be a separate axon tract (k) HuCD labelling only of (j) (l) Stage 22 The MLF labelled with DiO DiO was injected into therhombencephalon close to the ventral midline to labelMLF axonsMes mesencephalonMLFmedial longitudinal fascicle nMLF nucleus ofthe medial longitudinal fascicle op olfactory pit os optic stalk pros prosencephalon rh rhombencephalon tel telencephalon VC ventralcommissure


prosmes

MLF

rh

opIII

Tuj1

(a)

MLFIII

mes

rh

Tuj1

HuC

D

(b)

mes

rh

ep

MLF

(c)

nTPOC

op

(d)

Figure 2Development of axon tracts in the embryonic S canicula brain Lateral view ofwholemount embryonic brain Scale bars 100 120583m (a)Stage 23 overviewTheMLFneurones have projectedwell into the rhombencephalonThe scatteredMLFneurone population has increased insize (unfilled arrow)The first neurones were present in the dorsal prosencephalon (arrow) (b)ndash(d) Stage 23 double labelled with Tuj1 (green)and HuCD (red) (b) Neurones differentiated along the dorsal midline of the mesencephalon to give rise to the DTmesV (arrow) The MLFaxon tract was well formed (c)Three-four neurones appeared dorsally at the epiphysis (arrow) One neurone had projected its axon ventrallyalmost reaching theMLF (arrowhead)This axonwas a pioneer of theDVDT ScatteredMLFneuroneswere located rostrally to the initialMLFneurones (unfilled arrow) (d) Basal hypothalamus where the TPOC neurones (nTPOC) differentiated Scattered MLF neurones (unfilledarrow) ep epiphysis mes mesencephalon MLF medial longitudinal fascicle nTPOC nucleus of the tract of the postoptic commissure opolfactory pit pros prosencephalon III oculomotor nerve

telencephalon that will eventually give rise to the anteriordorsal telencephalic (ADt) population (Figure 2(a) arrow)

33 DetailedDescription of the Established Early Axon Scaffoldin the Embryonic S canicula Brain Between stages 23 and 25the early axon scaffold developed rapidlyThenumber of axontracts present increased and the early axon scaffold was verywell established and likely contained later follower axons thatwere using the scaffold for guidance (Figure 3(a)) From itsorigin close to the DMB the MLF extended caudally alongthe floor plate into the rhombencephalon (Figure 3(a)) TheDTmesV neurones projected axons from the dorsal midlineof the mesencephalon first ventrally then the axons turnedcaudally to pioneer the lateral longitudinal fascicle (LLF)that projected into the rhombencephalon (Figure 3(a)) Inthe rostral hypothalamus the TPOC neurones first projectedaxons dorsally while remaining ventral to the olfactory pitsbefore turning almost at a right angle to project caudallyonce the axons reached the SOT (Figure 3(c) arrowhead)Thedorsal telencephalon contained a largemixed population

of neurones the ADt (Figure 3(a)) These neurones appearedto be homologous to the dorsorostral cluster (drc) in zebrafish[16] and nPT in Xenopus [17] The ADt neurones projectedaxons to pioneer the AC in which axons crossed the anteriormidline dorsal to the olfactory pits The SOT axons alsoprojected from a population of neurones located withinthe ADt and projected axons ventrally to join the TPOC(Figure 3(b)) The tract of the habenular commissure (THC)projected axons from the telencephalon to the dorsal dien-cephalon to form the habenular commissure ([16] Figures3(a) and 3(b) Due to the density of neurones and axons atthis advanced stage it was difficult to determine the originof some of these later tracts An unknown axon tract (1) wasobserved projecting along a curved route rostral towards theMLF (Figures 3(a)ndash3(c)) It was unclear where the neuroneswere located but they appeared to be located ventrally in thehypothalamus (Figure 3(b) arrowhead) and projected axonsdorsally before turning along a more caudal route In thecaudal hypothalamus a population of neurones appeared ina fan-like shape and projected axons caudally into the MLFand were possibly homologous to the mammillotegmental


(a)

(b)

(c)

PCep

THCTPC

THC

TPOC

TPOC

TPOC

pros mes

SOT

SOT

SOTopV1

VC

AC

rh

ADt

DTmesV

LLFMLFIII

c

b

1

1

1

Tuj1

Figure 3 Description of the early axon scaffold at stage 25 in theembryonic S canicula brain Lateral view of whole mount embry-onic brain Scale bars 100 120583m (a) Overview of the early axon scaf-fold at stage 25 All axon tracts were well established Boxes indicatemagnified images in (b) and (c) (b) Prosencephalon The TPOCSOT and THC were well established An unidentified tract (1) waslocated caudally in the diencephalon The neurones were possiblylocated at the ventral midline (small arrowhead) (c) Unidentifiedtract 1 appeared to be projecting dorsally before turning to projectslightly caudally Possible MTT neurones were projecting axonsventrally (filled arrow) The TPOC axons have turned at a rightangle and project ventrally (arrowhead) AC anterior commissureADt anterior dorsal telencephalic neurones DTmesV descendingtract of the mesencephalic nucleus of the trigeminal nerve epepiphysis LLF lateral longitudinal fascicle mes mesencephalonMLF medial longitudinal fascicle op olfactory pit PC posteriorcommissure pros prosencephalon rh rhombencephalon SOTsupraoptic tract tel telencephalon THC tract of the habenularcommissure TPC tract of the posterior commissure TPOC tractof the postoptic commissure V1 ophthalmic profundus nerve VCventral commissure III oculomotor nerve

tract (MTT) (Figure 3(c) arrow) The tract of the posteriorcommissure (TPC) projected along the DMB forming the PCat the dorsal midline (Figure 3(a)) However it was unclearwhere the TPC neurones were located The location of theTPC along the DMB would suggest that the MLF neuroneswere both diencephalic and mesencephalic by stage 25 TheDVDT axon was likely to still be present at stage 25 howeverlabelling of the tract was not clear in the overview image(Figure 3(a)) The VC formed across the ventral midline atthe DMB with most axons present in the mesencephalon(Figure 3(a))

4 Discussion

We have presented a time series for the development ofthe initial axon tracts that form in the rostral S caniculaembryonic brain Eight main tracts were observed the MLFTPOC DVDT DTmesV TPC MTT SOT and THC as wellas three commissures the PC VC and AC (Figure 4(c))Thisbasic setup of the first axon tracts from a small number ofneurones in the embryonic brain was very similar to the earlyaxon scaffold characterised in other vertebrates Togetherwith similar studies in the lamprey [2] andmouse [4 6] theseresults confirm the ancient conserved scaffold that appears inthe vertebrate brain

41 Initial Neurones That Differentiated in the S caniculaBrain The MLF neurones were the first to appear in theembryonic brain at stage 18 (Figure 4(a))TheMLF remainedthe most prominent tract throughout much of the early axonscaffold development until stage 23 when other neuronesstarted to differentiate (Figure 4(b)) The appearance of theMLF neurones first is in line with many other vertebrates [1ndash5 18]

In the absence of an accurate description of themolecularand anatomical subdivisions of the embryonic cat sharkbrain the precise location of the neurones and their asso-ciated tracts was difficult to determine accurately Howeveras the TPC projects along the DMB in mouse [4] and chick[5] it was likely that the transversal projection of the TPCaxons in the diencephalonmesencephalon region of the Scanicula brain also aligns with this boundary (Figure 4(c))The location of the MLF neurones was more difficult todetermine as in chick and zebrafish the neurones are strictlydiencephalic [5 19] whereas in mouse the neurones are bothdiencephalic and mesencephalic [4] At later stages of Scanicula development the MLF neurones have been shownto be strictly within prosomere 1 [11]

42 Comparison of Early Axon Scaffold Formation in Scanicula with Other Vertebrates The early axon scaffoldof S canicula characterised here shares a number of keyfeatures with its counterpart in jawless (lamprey) and jawedvertebrates The development of the early scaffold starts withthe formation of the MLF as in most vertebrates Furthertracts like the TPOC TPC SOT and VC that could beidentified in the embryonic cat shark brain are found in allvertebrates [2] indicating that they represent structures of thelast common vertebrate ancestor

Along with these similarities there were also differencesA noticeable feature of the S canicula (and other cat sharkspecies) brain was the distinct brain vesicles including anenlarged telencephalon making its brain structure moresimilar to that of the amniotes rather than the anamniotesA possible reason for this could be that when the cat sharkhatches they are more developed as they have a longerdevelopmental period compared with anamniotes such aszebrafish or Xenopus and do not have a larval stage

One striking difference in axon tract development wasthe projection of the TPOC axons (Figure 4(c)) While theTPOC neurones were present in the hypothalamus like inother vertebrate species the TPOC axons do not contributeto the ventral longitudinal tract (VLT) in these early stagesIn chick the VLT is composed of the TPOC MTT andMLFthis feature is also highly conserved [5]While theTPOC tractformed from the nTPOC located in the rostral hypothalamusthe route the TPOC axons took in the shark was differentto that analysed in other vertebrates [4 5] By stage 25 atract homologous to the MTT was identified (Figure 4(c))These neurones appeared to be in the caudal hypothalamuswhere they were also located in chick and mouse [5] Theeventual shape of the neurone population was very differentforming a fan-like shape (Figure 4(c)) In the most rostralpart of the brain the POC formed from nTPOC neurones


Stage 18

pros

nMLF

mes

rh

os

(a)

Stage 23

ADt

op

tel

di

nTPOC

ep

DVDT

MLF

rh

DTmesV

mes

(b)

Stage 25

AC

ADt

op

TPOC

SOT

THC

tel

MTT

DVDT

di

ep

1

VC

MLF

rh

LLF

TPC

DTmesV

mes

(c)

Figure 4 Developmental series of the S canicula early axon scaffold Schematic representation with colour coding for the neurones andtheir associated axon tracts that arise in the S canicula brain (a) Stage 18 the appearance of the first neurones in the rostral brain (b) Stage23 MLF axons were projecting into the rhombencephalon and the nTPOC DTmesV and DVDT have appeared (c) Stage 25 the earlyaxon scaffold appeared to be well established with the addition of the AC SOT THC TPC VC and LLF AC anterior commissure ADtanterior dorsal telencephalic neurones di diencephalon DTmesV descending tract of themesencephalic nucleus of the trigeminal nerve epepiphysis LLF lateral longitudinal fascicle nMLF nucleus of the medial longitudinal fascicle nTPOC nucleus of the tract of the postopticcommissure mes mesencephalon MLF medial longitudinal fascicle MTT mammillotegmental tract op olfactory pit os optic stalk PCposterior commissure rh rhombencephalon SOT supraoptic tract tel telencephalon THC tract of the habenular commissure TPC tractof the posterior commissure TPOC tract of the postoptic commissure VC ventral commissure

and these axons cross the midline in anamniotes [1] ThePOC itself was not visible in the S canicula stages analysedhere but has previously been identified in the S torazamebrain [13] Compared with anamniotes the amniotes havefewer commissures during initial development although theamniotes eventually form these but at later stages Duringearly development S canicula forms the VC PC and AC(Figure 4(c)) While the VC and PC were also present inthe initial early axon scaffold in chick and mouse [4 5]the AC does not form until much later in chick [20] andmouse [21] In anamniotes the AC SOT and THC formfrom subpopulations within the large population of neuroneslocated in the telencephalon termed the ADt (Figure 4(c))No equivalent cluster of neurones is apparent in the earlyrostral brain of amniotes

In anamniotes theDTmesV formsmuch later in develop-ment [22 23] while interestingly in S canicula the DTmesV

formed in the mesencephalon early (Figures 4(b) and 4(c))reminiscent of amniotes [4 5] The DTmesV axons alsotook a slightly different route to that seen in the chick andmouse the DTmesV axons did not project as far ventralinto the mesencephalon alar plate in S canicula insteadthe axons projected along a caudal path to pioneer theLLF

The SOT formed during initial development of the earlyaxon scaffold in S canicula and other anamniotes but laterin other amniotes [24 25] The THC projects from theventral diencephalon to form the habenular commissure(Figure 4(c)) In zebrafish the THC appears around 28ndash30 hpf after initial formation of the early axon scaffoldadding further evidence that at stage 25 the S canicula earlyaxon scaffold was well established [16] The THC possiblycontained stria medullaris (SM) axons as well as these axonsfollow the same route [26]


In addition to the S torazame description [13] we identi-fied the VC MTT and an unidentified tract 1 (Figure 4(c))This unidentified tract 1 could be homologous to A13 neu-rones present in themouse [25] though tyrosine hydroxylaseimmunoreactivity has been reported to start at stage 26 [9 1127] while unidentified tract 1 was well visible already by stage25

43 Conclusions Our analysis highlights the conservation ofthe early axon scaffold as the first functional structure in theembryonic vertebrate brain The overall organisation of thecat shark axon tracts is similar to that described for othervertebrates including the early development of the mediallongitudinal fascicle Future work will involve lipophilic dyelabelling of individual tracts to determine where neuronalpopulations are located and the mapping of the neuronalclusters against themolecular pattern of the embryonic brainfor example the Pax6 expression domain marking the DMB[28]

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the funding of this studyby a studentship toMichelleWare by the Anatomical SocietyIn addition the study benefited from funding by the EUthrough the Interreg IVa 2 Seas programme ldquoTrans-ChannelNeuroscience Networkrdquo (TC2N)

References

[1] S W Wilson L S Ross T Parrett and S S Easter Jr ldquoThedevelopment of a simple scaffold of axon tracts in the brain ofthe embryonic zebrafish Brachydanio reriordquo Development vol108 no 1 pp 121ndash145 1990

[2] A Barreiro-Iglesias B Villar-Cheda X-M Abalo R Anadonand M C Rodicio ldquoThe early scaffold of axon tracts in thebrain of a primitive vertebrate the sea lampreyrdquo Brain ResearchBulletin vol 75 no 1 pp 42ndash52 2008

[3] V Hartenstein ldquoEarly pattern of neuronal differentiation inthe Xenopus embryonic brainstem and spinal cordrdquo Journal ofComparative Neurology vol 328 no 2 pp 213ndash231 1993

[4] G SMastick and S S Easter Jr ldquoInitial organization of neuronsand tracts in the embryonic mouse fore - and midbrainrdquoDevelopmental Biology vol 173 no 1 pp 79ndash94 1996

[5] M Ware and F R Schubert ldquoDevelopment of the early axonscaffold in the rostral brain of the chick embryordquo Journal ofAnatomy vol 219 no 2 pp 203ndash216 2011

[6] S S Easter Jr L S Ross and A Frankfurter ldquoInitial tractformation in the mouse brainrdquo Journal of Neuroscience vol 13no 1 pp 285ndash299 1993

[7] W J A J Smeets R Nieuwenhuys and B L Roberts TheCentral Nervous System of Cartilaginous Fishes Structure andFunctional Correlations Springer 1983

[8] R Anadon P Molist and I Rodriguez-Moldes ldquoDistributionof choline acetyltransferase immunoreactivity in the brain

of an elasmobranch the lesser spotted dogfish (Scyliorhinuscanicula)rdquo Journal of Comparative Neurology vol 420 no 2 pp139ndash170 2000

[9] I Carrera C Sueiro PMolist et al ldquoTemporal and spatial orga-nization of tyrosine hydroxylase-immunoreactive cell groupsin the embryonic brain of an elasmobranch the lesser-spotteddogfish Scyliorhinus caniculardquo Brain Research Bulletin vol 66no 4ndash6 pp 541ndash545 2005

[10] I Carrera P Molist R Anadon and I Rodrıguez-MoldesldquoDevelopment of the serotoninergic system in the central ner-vous system of a shark the lesser spotted dogfish Scyliorhinuscaniculardquo Journal of Comparative Neurology vol 511 no 6 pp804ndash831 2008

[11] S Ferreiro-Galve I Carrera E Candal et al ldquoThe segmentalorganization of the developing shark brain based on neu-rochemical markers with special attention to the prosen-cephalonrdquo Brain Research Bulletin vol 75 no 2ndash4 pp 236ndash2402008

[12] I Carrera R Anadon and I Rodrıguez-Moldes ldquoDevelop-ment of tyrosine hydroxylase-immunoreactive cell populationsand fiber pathways in the brain of the dogfish Scyliorhinuscanicula new perspectives on the evolution of the vertebratecatecholaminergic systemrdquo Journal of Comparative Neurologyvol 520 no 16 pp 3574ndash3603 2012

[13] S Kuratani and N Horigome ldquoDevelopmental morphology ofbranchiomeric nerves in a cat shark Scyliorhinus torazamewith special reference to rhombomeres cephalic mesodermand distribution patterns of cephalic crest cellsrdquo ZoologicalScience vol 17 no 7 pp 893ndash909 2000

[14] W W Ballard J Mellinger and H Lechenault ldquoA seriesof normal stages for development of Scyliorhinus canicula the lesser spotted dogfish (Chondrichthyes Scyliorhinidae)rdquoJournal of Experimental Zoology vol 267 pp 318ndash336 1993

[15] A Lumsden and R Keynes ldquoSegmental patterns of neuronaldevelopment in the chick hindbrainrdquoNature vol 337 no 6206pp 424ndash428 1989

[16] L S Ross T Parrett and S S Easter Jr ldquoAxonogenesis andmorphogenesis in the embryonic zebrafish brainrdquo Journal ofNeuroscience vol 12 no 2 pp 467ndash482 1992

[17] R B Anderson and B Key ldquoExpression of a novel N-CAMglycoform (NOC-1) on axon tracts in embryonic Xenopusbrainrdquo Developmental Dynamics vol 207 no 3 pp 263ndash2691996

[18] Y Ishikawa T Kage N Yamamoto et al ldquoAxonogenesis in themedaka embryonic brainrdquo Journal of Comparative Neurologyvol 476 no 3 pp 240ndash253 2004

[19] J T Hjorth and B Key ldquoAre pioneer axons guided by regulatorygene expression domains in the zebrafish forebrain High-resolution analysis of the patterning of the zebrafish brainduring axon tract formationrdquo Developmental Biology vol 229no 2 pp 271ndash286 2001

[20] S M Bardet J L E Ferran L Sanchez-Arrones and L PuellesldquoOntogenetic expression of sonic hedgehog in the chickensubpalliumrdquo Frontiers in Neuroanatomy 2010

[21] T Serafini S A Colamarino E D Leonardo et al ldquoNetrin-1is required for commissural axon guidance in the developingvertebrate nervous systemrdquo Cell vol 87 no 6 pp 1001ndash10141996

[22] C B Kimmel W K Metcalfe and E Schabtach ldquoT reticularinterneurons A class of serially repeating cells in the zebrafishhindbrainrdquo Journal of ComparativeNeurology vol 233 no 3 pp365ndash376 1985


[23] J J Kollros and M LThiesse ldquoGrowth and death of cells of themesencephalic fifth nucleus inXenopus laevis larvaerdquo Journal ofComparative Neurology vol 233 no 4 pp 481ndash489 1985

[24] H Ichijo and I Kawabata ldquoRoles of the telencephalic cells andtheir chondroitin sulfate proteoglycans in delimiting an anteriorborder of the retinal pathwayrdquo Journal of Neuroscience vol 21no 23 pp 9304ndash9314 2001

[25] H F Nural and G S Mastick ldquoPax6 guides a relay of pioneerlongitudinal axons in the embryonic mouse forebrainrdquo Journalof Comparative Neurology vol 479 no 4 pp 399ndash409 2004

[26] A Gaudin W Hofmeister and B Key ldquoChemoattractantaxon guidance cues regulate de novo axon trajectories in theembryonic forebrain of zebrafishrdquo Developmental Biology vol367 no 2 pp 126ndash139 2012

[27] I Rodrıguez-Moldes I Carrera S Pose-Mendez et al ldquoRegion-alization of the shark hindbrain a survey of an ancestralorganizationrdquo Frontiers in Neuroanatomy vol 5 article 16 2011

[28] Y Derobert B Baratte M Lepage and S Mazan ldquoPax6expression patterns in Lampetra fluviatilis and Scyliorhinuscanicula embryos suggest highly conserved roles in the earlyregionalization of the vertebrate brainrdquo Brain Research Bulletinvol 57 no 3-4 pp 277ndash280 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

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International Journal of

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Zoology


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BioinformaticsAdvances in

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Signal TransductionJournal of


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Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the formation at an older stage (Stage V) once the early axonscaffold appeared to be fully established [13]

The aim of this study was to characterise the early axonscaffold through a time series in the cat shark speciesScyliorhinus canicula using immunostaining

2 Materials and Methods

21 S canicula Embryos Sexually mature cat sharks (Scanicula) were caught locally in the summer off the coastof Portsmouth Hampshire UK and kept in a flow-through500 L seawater tank under ambient conditions at the Insti-tute of Marine Sciences University of Portsmouth Femalesregularly laid eggs wrapped around the outflow pipes of thetank S canicula eggs were incubated in flow-through sea-water at approximately 17∘C until they reached the requiredembryonic stage [14] To harvest the embryo a window wascut into the top egg to allow access The embryos werewashed in phosphate buffered saline (PBS) and fixed with4 paraformaldehydePBS or MEMFA (01M MOPS2mMEGTA1mM MgSO

437 formaldehyde 30ndash40 minutes

washed and stored in methanol)

22 Immunohistochemistry S canicula embryos were pre-pared for immunofluorescence by opening the rhomben-cephalon and the telencephalic vesicles The axon tractswere visualised using pan-neural antibodies Tuj1 mouse(Abcam ab7751 1 1000) or rabbit (Abcam ab18207 1 1000)and the neuronal populations specifically with anti-HuCD(Molecular Probes A21271 1 500) Primary antibodies werevisualised with Alexa fluorochrome-conjugated IgG anti-mouse (Invitrogen A11001 1 500) or anti-rabbit antibody(Invitrogen A11008 1 500) The protocol for immunohisto-chemistry has been described previously [15]

23 DiO Labeling Cyanine dye DiO (331015840-dioctade-cyloxacarbocyanine perchlorate Molecular Probes D275)was used to trace theMLF axons from the rhombencephalonS canicula embryonic brains were injected with dye aspreviously described [5]

24 Microscopy and Image Processing A Zeiss Stereo LumarV12 fluorescent stereomicroscope was used to obtain lowmagnification images of the embryos For more detailedimages Zeiss LSM 510 and LSM 710 confocal microscopeswere used Images were processed using Axiovision Rel 46ImageJ and Photoshop CS software

3 Results

S canicula embryos have been studied between stages 18 and25 [14] In terms of early axon development these stages wereequivalent to zebrafish stages 16 hours post fertilisation (hpf)to 24 hpfXenopus stages 22 to 32 chick stages HH11 toHH18and mouse stages E85 to E105 (data not published)

31 Detailed Description of the First Neurones to Differentiatein the Embryonic S canicula Brain Using an antibody against

120573III tubulin (Tuj1) the first neurones were detected withinthe S canicula embryonic brain at stage 18 (Figure 1(a) darrow) These neurones will form the nucleus of the MLF(nMLF) and occupied a location similar to theMLF neuronesin the chick brain [5] presumably rostral to the diencephalic-mesencephalic boundary (DMB) The MLF neurones startedprojecting axons caudally at stage 19 (Figure 1(b) e)TheMLFcontinued to project axons caudally at stage 20 (Figure 1(f))Also evident at stage 20 (Figure 1(f) unfilled arrow) and stage21 (Figure 1(g) unfilled arrow) were a scattered populationof neurones located more rostrally compared with the initialMLF neurones but still projecting axons into the MLFtract By stage 21 the MLF had formed a tight bundle thatprojected along the floor plate into the rhombencephalon(Figure 1(g) and data not shown) To confirm that the MLFhad reached the rhombencephalon the MLF axon tract waslabelled with DiO from the rhombencephalon at stage 22(Figure 1(c) orange spot) The dye diffused back to neuroneslocated in the diencephalon that belonged to the nMLF(Figure 1(l)) This confirmed that the axons of the MLFhad reached the rhombencephalon by stage 22 At stage 23the MLF was well established and appeared to be formedfrom three populations of neurones (Figures 1(h) 1(j) and1(k)) Double labelling with Tuj1 and HuCD revealed onepopulation thatwas located along the floor plate (Figures 1(h)1(j) and 1(k) arrow) another population that was locatedmore dorsally (Figures 1(j) and 1(k) unfilled arrowhead) andthe rostrally located scattered neurones (Figure 1(h) unfilledarrow)The population of neurones along the floor plate weretightly clustered and appeared to be contributing most ofthe axons to the MLF while the dorsal population was morescattered like the more rostral population There were axonsprojecting dorsally to the MLF axon tract and appeared to bea separate tract (Figure 2(j) arrowhead) These axons mostlikely projected from neurones that were part of the rostralMLF scattered population By stage 25 the MLF was wellestablished and the ventral commissure (VC) had formedacross the ventral midline (Figure 1(i))

32 Formation of the DTmesV DVDT and TPOC in theEmbryonic S canicula Brain Interestingly until stage 23 theMLF was still the only prominent tract in the S caniculabrain (Figures 1(a)ndash1(c)) Although it was not clear fromthe overview image at stage 23 (Figure 2(a)) higher mag-nification with double labelling of Tuj1 and HuCD anti-bodies revealed that there were other axon tracts beginningto form (Figures 2(b)ndash2(d)) Neurones appeared along thedorsal midline of the alar plate in the mesencephalon thatformed the nucleus of the DTmesV (nmesV) giving rise tothe DTmesV (Figure 2(b) arrow) There were 3-4 neuroneslocated dorsally at the epiphysis (Figure 2(c) arrow) Theseneurones started projecting axons ventrally pioneering thedorsoventral diencephalic tract (DVDT) One of these neu-rones had projected an axon ventrally almost reaching theMLF (Figure 2(c) arrowhead) The tract of the postopticcommissure (TPOC) formed from neurones located in therostral hypothalamus the nucleus of the TPOC (nTPOC)(Figure 3(d)) There were also neurones located in the dorsal


Stage 18

prosmes

dos

(a)

pros mese

Stage 19

nMLF

rh

(b)

pros mes

op

Stage 22

MLFrh

(c)

Stage 18

(d)

Stage 19

MLF

(e)

Stage 20

MLF

(f)

Stage 21

MLF

Tuj1

(g)

Stage 23

MLF

(h)

Stage 25

MLFVC

(i)

Stage 23

MLF

Tuj1

HuC

D

(j)

Stage 23

nMLF

(k)

Stage 22

MLF

DiO

(l)



prosmes

MLF

rh

opIII

Tuj1

(a)

MLFIII

mes

rh

Tuj1

HuC

D

(b)

mes

rh

ep

MLF

(c)

nTPOC

op

(d)






(a)

(b)

(c)

PCep

THCTPC

THC

TPOC

TPOC

TPOC

pros mes

SOT

SOT

SOTopV1

VC

AC

rh

ADt

DTmesV

LLFMLFIII

c

b

1

1

1

Tuj1



4 Discussion








Stage 18

pros

nMLF

mes

rh

os

(a)

Stage 23

ADt

op

tel

di

nTPOC

ep

DVDT

MLF

rh

DTmesV

mes

(b)

Stage 25

AC

ADt

op

TPOC

SOT

THC

tel

MTT

DVDT

di

ep

1

VC

MLF

rh

LLF

TPC

DTmesV

mes

(c)











Acknowledgments


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Stage 18

prosmes

dos

(a)

pros mese

Stage 19

nMLF

rh

(b)

pros mes

op

Stage 22

MLFrh

(c)

Stage 18

(d)

Stage 19

MLF

(e)

Stage 20

MLF

(f)

Stage 21

MLF

Tuj1

(g)

Stage 23

MLF

(h)

Stage 25

MLFVC

(i)

Stage 23

MLF

Tuj1

HuC

D

(j)

Stage 23

nMLF

(k)

Stage 22

MLF

DiO

(l)



prosmes

MLF

rh

opIII

Tuj1

(a)

MLFIII

mes

rh

Tuj1

HuC

D

(b)

mes

rh

ep

MLF

(c)

nTPOC

op

(d)






(a)

(b)

(c)

PCep

THCTPC

THC

TPOC

TPOC

TPOC

pros mes

SOT

SOT

SOTopV1

VC

AC

rh

ADt

DTmesV

LLFMLFIII

c

b

1

1

1

Tuj1



4 Discussion








Stage 18

pros

nMLF

mes

rh

os

(a)

Stage 23

ADt

op

tel

di

nTPOC

ep

DVDT

MLF

rh

DTmesV

mes

(b)

Stage 25

AC

ADt

op

TPOC

SOT

THC

tel

MTT

DVDT

di

ep

1

VC

MLF

rh

LLF

TPC

DTmesV

mes

(c)











Acknowledgments


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


prosmes

MLF

rh

opIII

Tuj1

(a)

MLFIII

mes

rh

Tuj1

HuC

D

(b)

mes

rh

ep

MLF

(c)

nTPOC

op

(d)






(a)

(b)

(c)

PCep

THCTPC

THC

TPOC

TPOC

TPOC

pros mes

SOT

SOT

SOTopV1

VC

AC

rh

ADt

DTmesV

LLFMLFIII

c

b

1

1

1

Tuj1



4 Discussion








Stage 18

pros

nMLF

mes

rh

os

(a)

Stage 23

ADt

op

tel

di

nTPOC

ep

DVDT

MLF

rh

DTmesV

mes

(b)

Stage 25

AC

ADt

op

TPOC

SOT

THC

tel

MTT

DVDT

di

ep

1

VC

MLF

rh

LLF

TPC

DTmesV

mes

(c)











Acknowledgments


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

(b)

(c)

PCep

THCTPC

THC

TPOC

TPOC

TPOC

pros mes

SOT

SOT

SOTopV1

VC

AC

rh

ADt

DTmesV

LLFMLFIII

c

b

1

1

1

Tuj1



4 Discussion








Stage 18

pros

nMLF

mes

rh

os

(a)

Stage 23

ADt

op

tel

di

nTPOC

ep

DVDT

MLF

rh

DTmesV

mes

(b)

Stage 25

AC

ADt

op

TPOC

SOT

THC

tel

MTT

DVDT

di

ep

1

VC

MLF

rh

LLF

TPC

DTmesV

mes

(c)











Acknowledgments


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Stage 18

pros

nMLF

mes

rh

os

(a)

Stage 23

ADt

op

tel

di

nTPOC

ep

DVDT

MLF

rh

DTmesV

mes

(b)

Stage 25

AC

ADt

op

TPOC

SOT

THC

tel

MTT

DVDT

di

ep

1

VC

MLF

rh

LLF

TPC

DTmesV

mes

(c)











Acknowledgments


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Acknowledgments


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Development of the Early Axon Scaffold in ...downloads.hindawi.com/archive/2014/196594.pdf · Research Article Development of the Early Axon Scaffold in the Rostral