ORIGINAL RESEARCH
published: 08 Ap il 2015
doi: 10.3389/ nana.2015.00037
Edi ed by:
Luis Puelles,
Uni e sidad de Mu cia, Spain
Re iewed by:
Manuel A. Pombal,
Uni e si y o Vigo, Spain
Alino Ma inez-Ma cos,
Uni e sidad de Cas illa, Spain
*Co espondence:
E a Candal,
Cen o de In es igaciones Biológicas,
Depa men o Cell Biology and
Ecology, Uni e si y o San iago de
Compos ela, Campus Vida, A enida
Lope Gómez de Ma zoa, s/n,
San iago de Compos ela E-15782,
Spain
[email p o ec ed]
Recei ed: 01 Decembe 2014
Pape pending published:
12 Janua y 2015
Accep ed: 09 Ma ch 2015
Published: 08 Ap il 2015
Ci a ion:
San os-Du án GN, Menue A,
Lagadec R, Mayeu H,
Fe ei o-Gal e S, Mazan S,
Rod íguez-Moldes I and Candal E
(2015) P osome ic o ganiza ion o he
hypo halamus in an elasmob anch,
he ca sha k Scylio hinus canicula.
F on . Neu oana . 9:37.
doi: 10.3389/ nana.2015.00037
P osome ic o ganiza ion o he
hypo halamus in an elasmob anch,
he ca sha k Scylio hinus canicula
Gab iel N. San os-Du án1,A naud Menue 2, Ronan Lagadec3,Hélène Mayeu 3,
Susana Fe ei o-Gal e3,Syl ie Mazan3,Isabel Rod íguez-Moldes1and E a Candal1*
1Cen o de In es igaciones Biológicas, Depa men o Cell Biology and Ecology, Uni e si y o San iago de Compos ela,
San iago de Compos ela, Spain, 2Cen e Na ional de la Reche che Scien i ique, Expe imen al and Molecula Immunology
and Neu ogene ics, Uni e si y o O leans, UMR7355, O leans, F ance, 3Cen e Na ional de la Reche che Scien i ique,
FR2424, De elopmen and E olu ion o Ve eb a es G oup, So bonne Uni e si és – Uni e si é Pie e e Ma ie Cu ie, Rosco ,
F ance
The hypo halamus has been a cen al opic in neu oana omy because o i s impo an
physiological unc ions, bu i s ma u e o ganiza ion emains elusi e. Deciphe ing i s
emb yonic and adul o ganiza ion is c ucial in an e olu iona y app oach o he
o ganiza ion o he e eb a e o eb ain. He e we s udied he molecula o ganiza ion
o he hypo halamus and neighbo ing elencephalic domains in a ca ilaginous
ish, he ca sha k, Scylio hinus canicula, ocusingonScFoxg1a,ScShh, ScNkx2.1,
ScDlx2/5,ScO p, and ScTb 1 exp ession p o iles and on he iden i ica ion α-
ace yla ed- ubulin-immuno eac i e (i ), TH-i , 5-HT-i , and GFAP-i s uc u es by means
o immunohis ochemis y. Analysis o he esul s wi hin he upda ed p osome ic model
amewo k suppo he exis ence o ala and basal his ogene ic compa men s in he
hypo halamus simila o hose desc ibed in he mouse, sugges ing he ances ali y
o hese subdi isions in jawed e eb a es. These da a p o ide new insigh s in o
hypo halamic o ganiza ion in ca ilaginous ishes and highligh he gene ali y o key
ea u es o he p osome ic model in jawed e eb a es.
Keywo ds: chond ich hyan, o eb ain pa e ning, e olu ion, de elopmen , p osome ic model, Shh,Nkx2.1, O p
In oduc ion
Biological di e si y eme ges, a leas in pa , h ough changes in de elopmen . O ganisms a e di -
e en because hei de elopmen al p ocess diffe and, wha is mo e, because hei de elopmen al
p ocess also e ol e (Ku sche a and Niklas, 2004;Mülle , 2007;Medina e al., 2011). Thus, he
Abb e ia ions: ABB, ala -basal bounda y; ac, an e io commissu e; AHy, ala hypo halamus; A , ac o e minal egion; AP,
ala pla e; BHy, basal hypo halamus; BP, basal pla e; CAHy, caudal pa o he ala hypo halamus; CBHy, caudal pa o
he basal hypo halamus; D, diencephalon; F, o eb ain; FP, floo pla e; HDB, hypo halamo-diencephalic bounda y; hp1, p o-
some e hp1 o peduncula hypo halamus; hp2, p osome e hp2 o e minal hypo halamus; IHB, in ahypo halamic bounda y;
MM, mammilla y a ea; MTT, mammillo- egmen al ac ; mz, ma ginal zone; os, op ic s alk; p1, p osome e 1; p2, p osome e
2; p2Tg, egmen al pa o p osome e 2; p3, p osome e 3; p3Tg, egmen al pa o p osome e 3; P, pallium; PM, pe imam-
milla y a ea; POA, p eop ic a ea; PPa, peduncula pa a en icula a ea; PRM, pe i e omammilla y a ea; PSPa, peduncula
subpa a en icula a ea; PThE, p e halamic eminence; RAHy, os al pa o he ala hypo halamus; RBHy, os al pa o he
basal hypo halamus; Rh, hombencephalon; RM, e omammilla y a ea; mc, e omammilla y commissu e; RP, oo pla e;
RTu, e o ube al domain; so , sup aop ic ac ; Sp, subpallium; T, elencephalon; TPa, e minal pa a en icula a ea; TPOC,
ac o he pos op ic commissu e; TSPa, e minal subpa a en icula a ea; Tu, ube al domain; z, en icula zone.
F on ie s in Neu oana omy | www. on ie sin.o g 1Ap il 2015 | Volume 9 | A icle 37
San os-Du án e al. P osome ic o ganiza ion o he ca sha k hypo halamus
unde s anding o he de elopmen o he e eb a e b ain
becomes undamen al o comp ehend i s s uc u e and e olu-
ion. In his con ex , he hypo halamus has been bo h a cen al
and elusi e opic. The hypo halamus is a conse ed in eg a-
i e cen e ha coo dina es au onomic, endoc ine, and limbic
esponses (Sa na and Ne sky, 1981;Kandel and Schwa z, 2001;
Bu le and Hodos, 2005).I sde elopmen ,a hebaseo he
e eb a e o eb ain (p osencephalon), in ol es complex pa -
e ning p ocesses dependen on diffe en signaling e en s ha
con e ge a his poin . I also unde goes a complex mo phological
de o ma ion du ing de elopmen , which misleads i s opologi-
cal ( s. opog aphic) loca ion (Shimamu a e al., 1995;Puelles
and Rubens ein, 2003;Puelles, 2009;Puelles e al., 2012). As
a esul , he hypo halamic o ganiza ion emains a ma e o
deba e (Figdo and S e n, 1993;Puelles and Rubens ein, 2003;
Shimogo i e al., 2010;Diez-Roux e al., 2011;Puelles e al.,
2012). C oss-species compa isons can be impo an o esol e
his issue, and an impo an effo o unde s and he unde -
lying uni y o hypo halamic emb yonic and adul o ganiza ion
ac oss e eb a es has been made ecen ly (Shimogo i e al., 2010;
Domínguez, 2011;Mo ales-Delgado e al., 2011, 2014;Mo eno
e al., 2012;Domínguez e al., 2013, 2014;He ge e al., 2014).
The p osome ic model (Puelles and Rubens ein, 2003;Puelles
e al., 2004, 2012;Medina, 2008;Puelles, 2009)hasbecomeakey
e e ence in such compa a i e s udies, since i offe s a mechanis-
ic pa adigm o he e eb a e b ain s uc u e and o ganiza ion.
Ini ially based on analyses o amnio es, his model defines o
he fi s ime ana omical s uc u es as de elopmen al hie a chical
uni s based on specifica ion mechanisms ha de e mine lon-
gi udinal and ans e se axis o ien a ion, segmen al s uc u e,
ansc ip ion ac o exp ession p ofiles and he eme gence o di -
e en ial his ogene ic domains (Puelles and Rubens ein, 2003;
Puelles, 2009;Ma ínez e al., 2012).
A majo in e es and no el y o his model is ha i pu s
emphasis on de elopmen al c i e ia (including opological ela-
ionships among ce ain mo phological landma ks, egula o y
gene exp ession pa e ns and signaling molecules). Tes ing hei
conse a ion ac oss e eb a es is a powe ul app oach o he
co ec es ablishmen o homologies be ween emb yonic e i o-
ies beyond amnio es (Puelles and Medina, 2002). The unde lying
no ion is ha o ma ion o he e eb a e b ain in ol es a con-
se ed co e o highly cons ained, in a ian mechanisms and
gene ic ne wo ks, which a e he basis o homology es ablish-
men . This in no way excludes he eme gence o di e sifica ions
h ough e olu ion, which a e he sou ce o he neu oana omic
di e si y obse ed among e eb a es.
La es upda es o he model p o ide no el iews on he
o ganiza ion o he os al-mos (seconda y) p osencephalon,
and i s elencephalic and hypo halamic moie ies (Puelles and
Rubens ein, 2003;Pombal e al., 2009;Puelles e al., 2012).
De ailed s udies in diffe en e eb a e g oups a e necessa y o
alida e he model assump ions. Ca ilaginous fishes o chon-
d ich hyans a e c ucial in his ask because hey a e among
he mos basal ex an g oups o gna hos omes (jawed e e-
b a es). Because o i s phylogene ic posi ion as he closes ou -
g oup o os eich hyans ( he o he majo phylum o gna hos-
omes, which includes bony fish and e apods), chond ich hyans
a e essen ial o econs uc gna hos ome ances al cha ac e is-
ics h ough compa isons wi h o he e eb a e models. He e we
s udied he molecula his ogene ic o ganiza ion o he hypo ha-
lamus and di ec ly adjoining e i o ies o an elasmob anch
ep esen a i e o one o he mos basal ex an gna hos ome lin-
eage, he ca sha k Scylio hinus canicula, and analyzed hem unde
he upda ed p osome ic amewo k. We ha e in eg a ed da a
om neu oepi helial specifica ion codes (based on he exp es-
sion o ca sha k o hologues o Foxg1a,Shh,Nkx2.1,Dlx2/5,O p,
and Tb 1), and om he dis ibu ion o α-ace yla ed- ubulin-
immuno eac i e (-i ) and TH-i cell g oups, neu on-fibe ac s
(5-HT-i ) and glial-p ocesses (GFAP-i ). In he sea ch o con-
se ed ai s among jawed e eb a es, we compa ed ou da a in
S. canicula wi h ha ob ained in mu ine models. Ou analysis
e eals a s ikingly high deg ee in he conse a ion o hypo ha-
lamic his ogene ic compa men s be ween chond ich hyan and
mu ine models. Fu he mo e, we iden ified some o he bound-
a ies and confi med some o he assump ions p edic ed by he
p osome ic model. Howe e , some diffe ences and disc epan-
cies also exis mainly conce ning he neu oepi helial specifica ion
gene ic codes o he basal hypo halamus (BHy). Simila s udies
a e equi ed in o he basal species o figu e ou i hese diffe ences
should p omp he model upda e o hey a e he consequence o
sha k specializa ion.
Ma e ials and Me hods
Phylogene ic Recons uc ions
Sequence alignmen s o he sequences lis ed in Table S1 we e
cons uc ed using he alignmen edi o Sea iew 3.0 and he
MUSCLE algo i hm. S. canicula sequences we e e ie ed by
blas n sea ches in ansc ip omic da abases ob ained by Sange
and Illumina sequencing o emb yonic and adul cDNA lib a ies
(s ages 8–25 and mixed adul issues). Maximum-likelihood ees
we e in e ed using he PhyML p og am e sion 3, he LG-
F+12+I subs i u ion model and he SPR algo i hm. Pos e io
p obabili ies (PPs) suppo ing g oupings we e calcula ed using
he aLRT algo i hm implemen ed in PhyML and a e displayed
as pe cen ages a he co esponding nodes. Only PP >80% a e
indica ed. The ees we e iewed and edi ed using Mega6.
Expe imen al Animals
Some emb yos o he ca sha k (lesse spo ed dogfish; S. canic-
ula) we e supplied by he Ma ine Biological Model Supply Se ice
o he CNRS UPMC Roscoff Biological S a ion (F ance) and
he Es ación de Bioloxía Ma iña da G aña (Galicia, Spain).
Addi ional emb yos we e kindly p o ided by he Aqua ia o
Gijón (As u ias, Spain), O G o e (Pon e ed a, Spain) and he
Aqua ium Finis e ae (A Co uña, Spain). Emb yos we e s aged
by hei ex e nal ea u es acco ding o Balla d e al. (1993). Fo
mo e in o ma ion abou he ela ionship o he emb yonic s ages
wi h body size, ges a ion and bi h, see Table 1 in Fe ei o-Gal e
e al. (2010). Thi y-se en emb yos om s ages 12 o 31 we e used
in his s udy. Eggs om diffe en b oods we e aised in seawa-
e anks in s anda d condi ions o empe a u e (15–16◦C), pH
(7.5–8.5) and salini y (35 g/L). Adequa e measu es we e aken o
F on ie s in Neu oana omy | www. on ie sin.o g 2Ap il 2015 | Volume 9 | A icle 37
San os-Du án e al. P osome ic o ganiza ion o he ca sha k hypo halamus
minimize animal pain o discom o . All p ocedu es con o med
o he guidelines es ablished by he Eu opean Communi ies
Council Di ec i e o 22 Sep embe 2010 (2010/63/UE) and by
he Spanish Royal Dec ee 53/2013 o animal expe imen a ion
and we e app o ed by he E hics Commi ee o he Uni e si y o
San iago de Compos ela.
Tissue P ocessing
Emb yos we e deeply anes he ized wi h 0.5% icaine me hane-
sul ona e (MS-222; Sigma, S . Louis, MO, USA) in seawa e and
sepa a ed om he yolk be o e fixa ion in 4% pa a o maldehyde
(PFA) in elasmob anch’s phospha e buffe [EPB: 0.1 M phospha e
buffe (PB) con aining 1,75% u ea, pH 7.4] o 48–72 h depend-
ing on he s age o de elopmen . Subsequen ly, hey we e insed
in phospha e buffe saline (PBS), c yop o ec ed wi h 30% suc ose
in PB, embedded in OCT compound (Tissue Tek, To ance, CA,
USA), and ozen wi h liquid ni ogen-cooled isopen ane. Pa allel
se ies o sec ions (12–20 μm hick) we e ob ained in ans e se
and sagi al planes on a c yos a and moun ed on Supe os Plus
(Menzel-Glasse , Madison, WI, USA) slides.
Single and Double Immunohis ochemis y
on Sec ions and Whole Moun s
Fo hea -induced epi ope e ie al, sec ions we e p e- ea ed
wi h 0.01 M ci a e buffe (pH 6.0) o 30 min a 95◦Cand
allowed o cool o 20–30 min a oom empe a u e (RT). Sec ions
we e hen insed wice in 0.05 M T is-buffe ed saline (TBS; pH
7.4) o 5 min each and incuba ed o e nigh wi h he p ima y
an ibody ( abbi an i-se o onin [an i-5-HT] polyclonal an i-
se um, DiaSo in, Immunos a , Hudson, WI, USA, dilu ed 1:5000;
polyclonal abbi an i-Sonic Hedgehog [an i-Shh], S a. C uz
Bio echnology, San a C uz, CA, USA, dilu ed 1:300; polyclonal
abbi an i-glial fib illa y acidic p o ein [an i-GFAP], Dako,
Glos up, Denma k, dilu ed 1:500; and monoclonal mouse an i-
y osine hyd oxilase [an i-TH], Millipo e, Bille ica, MA, USA,
dilu ed 1:500). App op ia e seconda y an ibodies [ho se adish
pe oxidase (HRP)-conjuga ed goa an i- abbi and an i-mouse,
BIORAD, dilu ed 1:200] we e incuba ed o 2 h a RT. Fo double
immunohis ochemis y (IHC) expe imen s, cock ails o p ima y
an ibodies we e mixed a op imal dilu ions and subsequen ly
de ec ed by using mix u es o app op ia e seconda y an ibod-
ies. Sec ions we e insed in dis illed wa e ( wice o 30 min),
allowed o d y o 2 h a 37◦Candmoun edinMOWIOL4-
88 Reagen (Calbiochem, Me kKGaA, Da ms ad , Ge many). All
dilu ions we e made wi h TBS con aining 15% donkey no mal
se um (DNS; Millipo e, Bille ica, MA, USA), 0.2% T i on X-100
(Sigma) and 2% bo ine se um albumin (BSA, Sigma). Double
IHC wi h p ima y an ibodies aised in he same species was
pe o med as desc ibed in To neha e e al. (2000).
Fo whole moun s emb yos we e p epa ed as p e iously
desc ibed in Ku a ani and Ho igome (2000) wi h mino modi-
fica ions. A e fixa ion wi h 4% PFA in 0.01 M PBS a 4◦C o
2 days, emb yos we e washed in 0.9% NaCl in dis illed wa e ,
dehyd a ed in g aded se ies o me hanol solu ions (50, 80, 100%)
ands o eda −20◦C. Samples o be s ained we e placed on ice
in 2 mL o dime hyl sul oxide (DMSO)/me hanol (1/1) un il
hey sank. Then, 0.5 mL o 10% T i on X-100/dis illed wa e was
added, and he emb yos we e incuba ed o 30 min a RT. A e
washing in 0.05 M TBS wi h 0.1% T i on X-100 (TST, pH 7.4)
he samples we e sequen ially blocked using spin-cla ified aque-
ous 1% pe iodic acid and 5% non- a d ied milk in TST (TSTM).
P ima y an ibody (monoclonal mouse an i-α-ace yla ed- ubulin,
Sigma, 1:1000) was dilu ed in TSTM con aining 0.1% sodium
azide o 2–4 days a RT wi h gen ly agi a ion on a shaking pla -
o m. The seconda y an ibody HRP-conjuga ed goa an i- abbi ,
BIORAD, dilu ion 1:200 in TSTM) was incuba ed o e nigh .
A e a final washing in TST, he emb yos we e p e-incuba ed
wi h 0.25 mg/mL diaminobenzidine e ahyd ochlo ide (DAB,
Sigma) in TST wi h 2.5 mg/mL nickel ammonium sul a e o 1 h,
and hen allowed o eac wi h DAB in TST con aining 2.5 mg/mL
nickel ammonium sul a e and 0.00075% H2O2 o 20–40 min a
RT. The eac ion was s opped using T is-HCL buffe ed saline
and specimens we e pos -fixed wi h 4% PFA o e nigh a 4◦C.
Epide mis and mesode mic de i a i es we e ca e ully emo ed
and specimens we e insed in g aded se ies o glyce ol (25, 50, 75,
and 100%) in o de o di ec ly obse e he neu al ube unde he
s e eomic oscope.
Con ols and Speci ici y o he An ibodies
No immunos aining was de ec ed when p ima y o seconda y
an ibodies we e omi ed du ing incuba ions. Con ols and speci-
fici y o an i-TH and an i-5-HT we e pe o med as desc ibed in
Pose-Méndez e al. (2014). The p ima y an i-α-ace yla ed- ubulin
an ibody has been shown o label ea ly diffe en ia ed neu ons and
hei p ocesses in he emb yonic ne ous sys em (Pipe no and
Fulle , 1985;Chi nis and Kuwada, 1990). The polyclonal an i-Shh
an ibody (San a C uz Bio echnology Inc, CA, USA) was aised in
abbi agains he amino acids 41–200 o Shh human p o ein. The
in si u hyb idiza ion (ISH) esul s we e simila o hose ob ained
by IHC, and he e o e alida e he specifici y o he an i-Shh
an ibody used he e.
In Si u Hyb idiza ion on Whole Moun
Emb yos and on Sec ions
We applied ISH o ScFoxg1a,ScShh (Compagnucci e al., 2013;
Quin ana-U zainqui, 2013), ScNkx2.1 (Quin ana-U zainqui
e al., 2012;Quin ana-U zainqui, 2013), ScDlx5 (Compagnucci
e al., 2013;Debiais-Thibaud e al., 2013), ScO p (Quin ana-
U zainqui, 2013), ScTb 1 (Quin ana-U zainqui, 2013), and
ScDlx2 (Quin ana-U zainqui e al., 2012;Compagnucci e al.,
2013;Debiais-Thibaud e al., 2013;Quin ana-U zainqui, 2013)
genes. These p obes we e selec ed om a collec ion o S. canicula
emb yonic cDNA lib a y (mixed s ages 9–22), cons uc ed in
pSPORT1,andsubmi ed ohigh h oughpu ESTsequencing.
cDNA agmen s we e cloned in pSPORT ec o s. Sense and
an isense digoxigenin-UTP-labeled and fluo escein-UTP-labeled
p obes we e syn hesized di ec ly by in i o ansc ip ion using
as empla es linea ized ecombinan plasmid DNA o cDNA
agmen s p epa ed by PCR amplifica ion o he ecombi-
nan plasmids. ISH in whole moun and on c yos a sec ions
was ca ied ou ollowing s anda d p o ocols (Coolen e al.,
2009). B iefly, sec ions we e pe meabilized wi h p o einase K,
hyb idized wi h sense o an isense p obes o e nigh a 65◦C(in
sec ions) o 70◦C (whole moun ) and incuba ed wi h he alkaline
F on ie s in Neu oana omy | www. on ie sin.o g 3Ap il 2015 | Volume 9 | A icle 37
San os-Du án e al. P osome ic o ganiza ion o he ca sha k hypo halamus
phospha ase-coupled an i-digoxigenin and an i-fluo escein
an ibody (1:2000, Roche Applied Science, Manheim, Ge many)
o e nigh a 4◦C. The colo eac ion was pe o med in he
p esence o BM-Pu ple (Roche). Con ol sense p obes did no
p oduce any de ec able signal.
Inhibi ion o he Shh Pa hway
Inhibi ion o he Shh pa hway was pe o med by in o o injec-
ion o he pha macological inhibi o cyclopamine in o de o es
whe he , as in os eich hyans, he ini ia ion o ScNkx2.1 exp ession
in he o eb ain is dependen on Shh. Fi s , 200 μLo asolu ion
con aining 1x PBS, 500 μMcyclopamineand5%DMSOwe e
injec ed h ough he shell o s age 15–16 S. canicula eggs. This
solu ion was eplaced by he same olume o 5% DMSO in 1x
PBS o con ol emb yos. The eggs we e main ained o 3 days in
oxygena ed sea wa e a 17◦C, wi h iabili ies highe han 90%.
Emb yos eached s age 18 in hese condi ions. They we e dis-
sec ed, fixed in PFA 4%, dehyd a ed and s o ed in me hanol 100%
p io o ISH.
Image Acquisi ion and Analysis
Ligh field images we e ob ained wi h an Olympus BX51 mic o-
scope equipped wi h an Olympus DP71 colo digi al came a.
In o o emb yos we e analyzed in he Olympus SZX12 s e e-
omic oscope fi ed o an Olympus DP12 colo digi al came a.
Pho og aphs we e adjus ed o b igh ness and con as and pla es
we e p epa ed using Adobe Pho oshop CS4 (Adobe, San Jose,
CA, USA).
Resul s
Iden i ica ion o Ca sha k O hologues o
he Genes S udied
Exhaus i e phylogene ic cha ac e iza ions o he ca sha k Dlx
gene epe oi e ha e been p e iously published (Debiais-Thibaud
e al., 2013), confi ming he iden i y o ScDlx2 and ScDlx5.In
o de o unambiguously iden i y he ca sha k o hologues o
Foxg1,Shh,Nkx2.1,O p, and Tb 1, we conduc ed sys ema ic
phylogene ic analyses o he co esponding e eb a e gene ami-
lies, including all he e eb a e classes de i ed om duplica ions
o a single ances al cho da e o hologue (Figu e 1). In each
case, phylogenies we e cons uc ed om alignmen s con ain-
ing deduced amino acid sequences o all pa alogous sequences
e ie ed om ca sha k ansc ip omic da abases and om a
ep esen a i e sampling o ac inop e ygians and sa cop e ygians.
The ees we e oo ed using a B anchios oma lo idae sequence,
excep in he case o O p which could no be ound in he
amphioxus Ensembl da abase. In he case o Foxg1, h ee s ongly
suppo ed classes (pos e io p obabili y o PP >90%), each
con aining a ca sha k and se e al os eich hyan sequences, we e
e ie ed, highligh ing o he fi s ime he p esence o h ee
gna hos ome Foxg1 classes (Figu e 1A). These classes we e
e med Foxg1a, Foxg1b, and Foxg1c, espec i ely. One coela-
can h and se e al ac inop e ygian sequences, bu no amphibian
o amnio e sequence, we e ound in he Foxg1b and Foxg1c
classes, sugges ing a loss o hei ep esen a i es in e apods.
We ocused he exp ession analysis on ScFoxg1a, he ca sha k
FIGURE 1 | Phylogene ic analysis o he Scylio hinus canicula genes
analyzed in his s udy. Phylogene ic ees o he Foxg1, Hedgehog,
Nkx2.1/Nkx2.4, O p and Tb 1/Tbx21/Eomes amilies a e shown in (A–E)
espec i ely. The numbe o subs i u ions pe si e is indica ed a he
bo om o each ee, on he le . S. canicula genes a e displayed in
ed. Abb e ia ions used: Hs, Homo sapiens (human); Gg, Gallus gallus
(chick); Ac, Anolis ca olinensis (anole liza d); X , Xenopus opicalis
(A ican clawed og); Lc, La ime ia chalumnae (coelacan h); Lo,
Lepisos eus ocula us (spo ed ga ); Ol, O yzias la ipes (medaka); D ,
Danio e io (zeb a ish); Cm, Callo hinchus milii (elephan sha k); Sc,
Scylio hinus canicula (ca sha k o lesse spo ed dog ish); B ,
B anchios oma lo idae (amphioxus).
F on ie s in Neu oana omy | www. on ie sin.o g 4Ap il 2015 | Volume 9 | A icle 37
San os-Du án e al. P osome ic o ganiza ion o he ca sha k hypo halamus
o hologue o he only Foxg1 gene e ained in all majo gna hos-
ome lineages including e apods. The ee opology ob ained
o he Hedgehog amily confi med he p esence o he h ee
gna hos ome classes, co esponding o he Indian Hedgehog,
Dese Hedgehog and Sonic Hedgehog classes al eady epo ed
in os eich hyans, and confi med ScShh as he ep esen a i e o he
la e (Figu e 1B). Conce ning he Nkx2.1/Nkx2.4 amily, a sin-
gle ca sha k gene could be iden ified and i was unambiguously
assigned o he Nkx2.1 class based on he s ongly suppo ed
g ouping o i s deduced amino acid sequence wi h eleos , chick,
and human Nkx2.1 sequences (PP =97%; Figu e 1C). This
gene is he e o e e e ed o as ScNkx2.1 he ea e . A single ca -
sha k O p ela ed sequence, e med ScO p, could be ound and
as expec ed, i clus e ed wi h he elephan sha k sequence anno-
a ed as O p in he econs uc ion shown in Figu e 1D. Finally,
he Tb 1, Tbx21, and Eomes classes we e e ie ed wi h high
s a is ical suppo (PP =83, 100, and 99%, espec i ely) wi hin
he Tb 1/Tbx21/Eomes amily. Each class con ained a single
ca sha k sequence a he expec ed posi ion, allowing an unam-
biguous iden ifica ion o he ScTb 1 gene analyzed in his s udy
(Figu e 1E).
P elimina Conside a ions Conce ning
Ve eb a e Segmen al P osencephalic
O ganiza ion
The o ganiza ion o he sha k hypo halamus has been analyzed
in he amewo k o he upda ed p osome ic model (Puelles
e al., 2012). Figu e 2 summa izes he gene al a chi ec u e o
he hypo halamus in mouse acco ding o he upda ed p o-
some ic model (Puelles e al., 2012). This model is mainly
inspi ed in mu ine da a hough i is usually assumed ha i
can be ex apola ed o all e eb a es because i also in eg a es
in o ma ion om o he e eb a es (Puelles and Rubens ein,
2003;Pombal e al., 2009;Puelles, 2009). Indeed, his model
ep esen s a use ul de elopmen al and compa a i e amewo k
since i makes use o concep s, nomencla u e and opolog-
ical e e ences ha can be used ac oss diffe en e eb a e
species.
The p osome ic model es ablishes ha hypo halamus and
elencephalon a e pa o he seconda y p osencephalon, which
is unde s ood as a segmen al uni a he os al-mos poin o
he neu al ube, he hypo halamus being loca ed en al o he
elencephalon and os al o he diencephalon (see Figu e 2A).
The model also pos ula es ha he os al-mos poin o he
b ain, e e ed as he ac o e minal egion (A ), lies a he os al
bo de o he seconda y p osencephalon. This egion is es ic ed
o he on al bo de o he neu al ube whe e le and igh ala
and basal pla es mee . This bo de expands do so- en ally om
he os al-mos oo pla e (which is elencephalic) o he os al-
mos floo pla e (which is hypo halamic). Thus, e e y s uc u e
classically conside ed being do sal o en al o hese poin s
(see a owheads in Figu e 2A), should be conside ed as cau-
dal in his amewo k. O no e, he an e io commissu e, loca ed
in he os al-mos oo pla e, is a clea landma k o bo h he
do so- en al and os o-caudal axis (Puelles e al., 2012;seealso
Figu es 2A,B).
FIGURE 2 | Squema ic ep esen a ions o he p osencephalon o ea ly
(A) and la e (B,C) mouse emb yo o show co espondence o
longi udinal and an e se domains in he seconda y p osencephalon
unde he upda ed p osome ic model. Domains in (A) a e illus a ed
acco ding o Figu e 1.1C in Ma ínez e al. (2012). Domains in (B,C) a e
illus a ed acco ding o Figu e 8.5B in Puelles e al. (2012). (A) Longi udinal
domains in ea ly emb yos. The a owheads ma k bo h he do so-caudal and
en o-caudal limi s o he ac o e minal e i o y (A ). This e i o y is conside ed
he os al-mos domain o he neu al ube. The do so-caudal limi o he A can
be iden i ied caudal o he an e io commissu e. (B) Longi udinal and ans e se
o ganiza ion in la e emb yos. (C) Segmen al o ganiza ion o he seconda y
p osencephalon acco ding o he p osome ic model. Fo abb e ia ions, see lis .
F on ie s in Neu oana omy | www. on ie sin.o g 5Ap il 2015 | Volume 9 | A icle 37
San os-Du án e al. P osome ic o ganiza ion o he ca sha k hypo halamus
The seconda y p osencephalon p esen s wo ue segmen s
os o-caudally a anged (Figu e 2B): hp2 ( os al o e minal)
and hp1 (caudal o peduncula ). Each segmen ha bo s elen-
cephalic and hypo halamic de i a i es (Figu es 2B,C). Howe e ,
he elencephalon ha bo s only oo and ala pla es while he
hypo halamus ha bo s ala , basal, and floo pla e de i a i es. The
exis ence o hese segmen s is suppo ed by se e al genes di -
e en ially exp essed in he os o-caudal axis, he loca ion o
commissu es in he oo and floo pla es (an e io and e omam-
milla y commissu es, espec i ely), and he cou se o impo an
ac s [medial o eb ain bundle (m b); la e al o eb ain bundle
(l b); and o nix ( x)] unning by a common pa h a he os al
bo de o hp1, h ough ala and basal pla es. These da a, in u n,
suppo he exis ence o an in e segmen al bounda y ha sepa-
a es e minal and peduncula subdi isions o bo h elencephalon
and hypo halamus, which is e e ed as he in ahypo halamic
bounda y (IHB; Figu es 2B,C). Caudally, he seconda y p os-
encephalon limi s wi h he diencephalon a he hypo halamic
diencephalic bo de (HDB), ano he in e segmen al limi among
hp1 and p3, hough i should be no iced ha pa o he caudal
limi o he seconda y p osencephalon does co espond o he
elencephalon (Puelles e al., 2012;seealsoFigu e 2C).
The model conside s he adul hypo halamic o ganiza ion
a anged in diffe en his ogene ic e i o ies defined by neu oep-
i helial specifica ion codes and adial uni s (Puelles and Medina,
2002;Puelles e al., 2012). These codes e eal ha elencephalon
and hypo halamus belong o diffe en his ogene ic e i o ies
being he p eop ic a ea (POA) he unique e minal e i o y o
he elencephalon (Figu e 2C). O no e, he POA also ha bo s he
an e io commissu e (Puelles e al., 2012;seealsoFigu es 2B,C).
ScFoxg1a Exp ession
In mice, Foxg1 is one o he ea lies ansc ip ion ac o s
exp essed specifically in he pa o he neu al pla e ha gi es
ise o he elencephalon and i emains exp essed h oughou he
elencephalon du ing emb yonic de elopmen (see Manuel e al.,
2011). In an a emp o disc imina e elencephalic and unde ly-
ing hypo halamic domains h oughou S. canicula de elopmen ,
we ha e analyzed he exp ession o ScFoxg1a in he de eloping
ne oussys emo hisspecies.A s age18,ScFoxg1a exp ession
was ound in he do sal-mos po ion o he seconda y p osen-
cephalon including he op ic cup, ex ending om he le el o he
op ic s alk (which is loca ed os ally, wi hin he A ) up o a cau-
dal poin in he oo pla e, which has been en a i ely iden ified
as he do sal bo de be ween he elencephalon and he dien-
cephalon (Figu e 3A). A s age 22, ScFoxg1a was obse ed in he
elencephalon and in he nasal pa o he op ic cup (Figu e 3B).
The exp ession in he elencephalon was main ained un il la e
s ages o de elopmen (Figu e 3C), which allowed iden i ying he
bo de be ween he elencephalon and he hypo halamus.
ScShh Exp ession
ScShh exp ession was de ec ed du ing gas ula ion (s age 12) in
he caudal midline o he emb yo (da a no shown). A s age
14, du ing ea ly neu ula ion, i has been de ec ed in he axial
mesode m o he no ocho d and he p echo dal pla e and in he
ec ode m o he caudal midline (da a no shown). A e he clo-
su e o he neu al ube (s age 17), he signal was de ec ed as a
en al longi udinal con inuous band ha ex ends om he cau-
dal end o he spinal co d o he A o he o eb ain, oughly
a he le el o he op ic s alk (Figu e 3D). As in o he e e-
b a es (Shimamu a e al., 1995), he exp ession o ScShh can be
used o define he ala -basal bounda y (ABB; Figu es 3E–H). A
s age 19, ScShh exp ession became down egula ed in he o e-
b ain o p og essi ely gi e ise o a caudal and a os al domain
(a ow in Figu es 3E–H). The na ow ans e se and do sally
di ec ed s ipe o ScShh-exp essing cells wi hin he caudal domain
was iden ified as he de eloping zona limi ans in a halamica (zli;
a owhead in Figu e 3G). The os al bo de o he ScShh cau-
dal domain, in u n, was somewha ex ended os al o he HDB
(Puelles e al., 2012), which a his s age was iden ified as he poin
whe e he neu al ube expands o acqui e he dis inc i e shape
o he en al hypo halamus. The e o e, he BHy appea ed o be
di ided in h ee domains: wo posi i e o ScShh (one os al and
o he caudal) and one (in e media e) nega i e o ScShh (a ow
in Figu es 3G,H;seealsoFigu e5HinCompagnucci e al., 2013).
O no e, he do sal bo de o he os al domain (p esumably co -
esponding o he ABB) seems o codis ibu e wi h α-ace yla ed-
ubulin-immuno eac i e (-i ) longi udinal ac s (a owheads in
Figu e 3I). A s age 24 (Figu e 3H), a new domain eme ged
wi hin he elencephalon. This sho domain (a owhead in
Figu e 3H) ex ended om a egion loca ed do sally o he op ic
s alk wi hou eaching he p ospec i e e i o y o he an e-
io commissu e ( ha can be iden ified a ea ly de elopmen by
means o α- ubulin-immuno eac i i y; as e isk in Figu es 3H,I).
A clea gap o exp ession was obse ed be ween his elencephalic
domain and he os al hypo halamic one (Figu e 3H). The elen-
cephalic domain was loca ed medially while he hypo halamic
one also expanded la e ally (no shown). F om s age 27 onwa d
he zli expanded do sally owa d he oo pla e (a owhead in
Figu e 4A). A s age 29 he medio-la e al his ologic o ganiza ion
o he de eloping walls o he o eb ain become mo e e iden .
As in p e ious de elopmen al s ages, Shh immuno eac i i y was
clea ly iden ified in he basal pla e o he diencephalon en e ing
he caudo- en al pa o he BHy (a ow in Figu es 4A,B,B)
and in he os o-do sal pa o he BHy (Figu es 4A,B), so ha a
clea nega i e gap o Shh-immuno eac i i y occupied mos o he
caudal BHy (CBHy; Figu es 4A,B) and pa o he os al BHy
(RBHy). In he elencephalon, Shh-immuno eac i i y expanded
caudally beyond he p ospec i e e i o y o he an e io com-
missu e (a owhead in Figu e 4B; compa e wi h Figu e 3H). O
no e, om la e s age 30 onwa d, Shh-immuno eac i i y is down-
egula ed in he CBHy and basal diencephalon, excep in he zli
(da a no shown).
ScNkx2.1 Exp ession
The exp ession o ScNkx2.1 was fi s de ec ed a s age 18 in
he os o- en al po ion o he o eb ain, in a longi udi-
nal band which ex ended en al o he op ic s alk (a ow-
head in Figu e 3J). A s age 23 ScNkx2.1 was exp essed
in mos o he BHy (Figu e 3K). Diffe en ly om ScShh,
ScNkx2.1 delimi ed he ABB e en in he CBHy (Figu e 3K;
F on ie s in Neu oana omy | www. on ie sin.o g 6Ap il 2015 | Volume 9 | A icle 37
San os-Du án e al. P osome ic o ganiza ion o he ca sha k hypo halamus
FIGURE 3 | Con inued
F on ie s in Neu oana omy | www. on ie sin.o g 7Ap il 2015 | Volume 9 | A icle 37
San os-Du án e al. P osome ic o ganiza ion o he ca sha k hypo halamus
FIGURE 3 |Con inued
Regionaliza ion o he hypo halamus and neighbo e i o ies in emb yos o S. canicula om s ages 18–29 based on he exp ession pa e n o
ScFoxg1a (A–C), ScShh (D–H), ScNkx2.1 (J–L), ScDlx5 (M–O), ScO p (P,Q), ScTb 1 (R) genes and α-ace yla ed- ubulin immuno eac i i y (I). In all panels,
do ed lines de ine he hypo halamo- elencephalic bounda y (HTB), dashed lines indica e he caudal bo de o he seconda y p osencephalon and ed lines indica e
he ABB. (A–C) ScFoxg1a exp ession in he seconda y p osencephalon a indica ed s ages. The a owheads in (A) ma k he caudo-do sal and os o- en al limi
o ScFoxg1a exp ession. (D–H) ScShh exp ession a he indica ed s ages. The a owhead in (D) ma ks he os al-mos poin o ScShh exp ession in he o eb ain.
The a ows in (E–H) indica e he down egula ion o ScShh exp ession in he hypo halamus. The a owhead in (G) poin s o he de eloping zli. The a owhead in
(H) poin s o a no el domain in he elencephalon. The as e isk in (H) ma ks he p ospec i e e i o y o he an e io commissu e. (I) An i-α-ace yla ed- ubulin IHC
o show h ee se s o ac s a s age 25. These ac s a e classically e e ed as so , TPOC and MTT. The as e isk indica es he e i o y o he de eloping an e io
commissu e. The a owheads poin o he longi udinal TPOC. The a ow poin s o he os al-mos ex ension o he MTT. (J–L) ScNkx2.1 exp ession a he indica ed
s ages. The a owhead in (J) poin s o he os al-mos poin o ScNkx2.1 exp ession a s age 18, which was es ic ed o a sho longi udinal domain en ally o
he op ic s alk. The a ow in (K,L) poin s o a small ScNkx2.1-nega i e domain a he mos caudo- en al BHy. The as e isk in (K,L) ma ks he p ospec i e e i o y
o he an e io commissu e. The a owhead in (K,L) poin s o a domain in he elencephalon ha sp ead os o-caudally. (M–O) ScDlx5 exp ession a he indica ed
s ages. The a owheads in (M,N) indica e ScDlx5 exp ession in he ol ac o y placode and he an e io pa o he elencephalon. The as e isk in (N) indica es he
p ospec i e e i o y o he an e io commissu e. The a owheads in (O) poin o he en al and caudal expansion o ScDlx5 exp ession in he elencephalon. This
domain was ai ly con inuous wi h a longi udinal band o ScDlx5 o e he ABB. The a ows in (O) poin o ScDlx5-exp essing domains ha sp ead in o he BHy. (P,Q)
ScO p exp ession a he indica ed s ages. The a owhead in (P) indica es a es ic ed domain o ScO p exp ession en ally loca ed wi h espec o he op ic s alk.
The exp ession o ScO p in he hypo halamus was ain compa ed o ha o he Rh. The whi e a owhead in (Q) poin s o ScO p exp ession in he AHy. Two addi ional
ScO p-exp essing domains we e obse ed in he BHy. (R) ScTb 1 exp ession a s age 25 was ound in pa o he elencephalon and a he do sal-mos pa o he
os al diencephalon (whi e a owhead). The as e isk indica es he p ospec i e e i o y o he an e io commissu e. Fo abb e ia ions, see lis .
compa e wi h Figu e 3G), hough a small gap o exp es-
sion was obse ed wi hin he caudo- en al pa o he BHy
(a ow in Figu e 3K). A second domain eme ged in he
elencephalon a his s age (a owhead in Figu e 3K). This
domain was es ic ed o he os al-mos po ion o he elen-
cephalon and ex ended om a egion loca ed do sally o he
op ic s alk o he p ospec i e e i o y o he an e io com-
missu e (as e isk in Figu e 3K). A clea gap o exp ession
was obse ed be ween he elencephalic and he hypo ha-
lamic domains. A s age 25 (Figu e 3L), as in p e ious
de elopmen al s ages, ScNkx2.1 exp ession was lacking in a
small domain loca ed wi hin he caudo- en al BHy (a ow in
Figu e 3L). This egion seems o fi wi h he os al bo de o
a basal α-ace yla ed- ubulin-i ac [see mammillo- egmen al
ac (MTT) in Figu e 3I]. In he elencephalon, ScNkx2.1
exp ession became caudally expanded beyond he p ospec i e
e i o y o he an e io commissu e (as e isk in Figu e 3L).
A s age 29, as in p e ious de elopmen al s ages, ScNkx2.1
was obse ed h oughou mos o he BHy, excep in a small
wedge-shaped domain wi hin he caudo- en al BHy (a ow-
head in Figu es 4C,D). G oups o ScNkx2.1-exp essing cells in
he caudo- en al po ion o he hypo halamus we e obse ed
along he ma ginal zone (a ows in Figu es 4C,C,D). In he
elencephalon, ScNkx2.1 exp ession expanded beyond he e -
i o y i occupied a p e ious de elopmen al s ages (as e isk
in Figu e 4D). O no e, Shh immuno eac i i y was o e lap-
ping wi h ScNkx2.1 exp ession beyond he an e io commissu e
(Figu e 4D).
In o de o es whe he , as in os eich hyans, he ini ia ion
o ScNkx2.1 exp ession in he o eb ain is dependen on Shh,
we used in o o injec ions o he Shh inhibi o cyclopamine. All
con ol emb yos (n=4) exhibi ed he expec ed ScNkx2.1sig-
nal in he os al-mos and en al-mos po ion o he o eb ain
(Figu e 5). This signal was los in all emb yos dissec ed ollowing
cyclopamine ea men (n=3), suppo ing he conclusion ha
Shh signaling is equi ed o he ini ia ion o ScNkx2.1 exp ession
in S. canicula as in os eich hyans.
ScDlx2/ScDlx5 Exp ession
We analyzed he exp ession o ScDlx5 om s age 18 onwa d and
he exp ession o ScDlx2 om s age 29 onwa d. Fai ly iden ical
esul s we e obse ed wi h bo h ma ke s in he b ain o S. canic-
ula om s age 29 onwa d, so we use ScDlx2/5 a hese s ages o
e e indis inc ly o bo h.
Gene al ea u es o ScDlx5 exp ession and de ailed p ofiles in
he de eloping b anchial a ches ha e been p e iously desc ibed
om s age 15 o s age 27 in Compagnucci e al. (2013)and
om s age 15 o s age 25 in Debiais-Thibaud e al. (2013). We
e isi ed hese da a ocusing on he de eloping o eb ain. A
s age 18, ScDlx5 exp ession was ound in he mos an e io pa
o he neu al ube (Figu e 3M; compa e wi h Figu e 3A;see
also Figu e 5C1 in Debiais-Thibaud e al., 2013). F om s age
21 o 25, ScDlx5 becomes mos ly es ic ed o he an e io -
mos pa o he elencephalon and o he ol ac o y placodes
(Figu e 3N;seealsoFigu e4GinCompagnucci e al., 2013
and Figu e 5C’1 in Debiais-Thibaud e al., 2013). Howe e ,
a la e s ages (Figu e 3O), ScDlx5 exp ession sp ead cau-
dally and en ally (a owheads in Figu e 3O) and eached he
os al-mos po ion o he op ic s alk (see also Figu e 9C in
Debiais-Thibaud e al., 2013). This domain was ai ly con in-
uous wi h a longi udinal band o ScDlx5 ha c ossed h ough
he hypo halamus o e he ABB (compa e wi h Figu es 3H,L)
and en e ed p3 (Figu e 3O). The e o e, his domain delinea es
he ABB along he hypo halamus. O no e, he longi udinal
domain appea ed o codis ibu e wi h α-ace yla ed- ubulin ac s
(a owheads in Figu e 3I). Al hough bo h ScDlx5 domains we e
con inuous, a wedge-shaped a ea o educed signal in ensi y
was obse ed be ween he do sal ( elencephalic) and he en al
(hypo halamic-diencephalic) domains (Figu e 3O). Two bands
o cells we e addi ionally obse ed, which we e en ally loca ed
wi h espec o he longi udinal domain (a ows in Figu e 3O).
One was loca ed a i s caudal end and sp ead en al-wa d a
he os al end o p3. The o he sp ead pe pendicula ly o he
ABB om he caudo-do sal pa o he BHy up o he os-
al hypo halamus (Figu e 3O). O no e, his ScDlx5-exp essing
F on ie s in Neu oana omy | www. on ie sin.o g 8Ap il 2015 | Volume 9 | A icle 37
San os-Du án e al. P osome ic o ganiza ion o he ca sha k hypo halamus
FIGURE 4 | Con inued
F on ie s in Neu oana omy | www. on ie sin.o g 9Ap il 2015 | Volume 9 | A icle 37
San os-Du án e al. P osome ic o ganiza ion o he ca sha k hypo halamus
Région Cen e, Région B e agne (EVOVERT g an num-
be 049755; PEPTISAN p ojec ), Na ional Resea ch Agency
(g an ANR-09-BLAN-026201), CNRS, Uni e si é d’O léans and
Uni e si é Pie e e Ma ie Cu ie. GNSD would like o hank
Spanish SEPE o i s unding suppo .
Supplemen a y Ma e ial
The Supplemen a y Ma e ial o his a icle can be ound online
a : h p://www. on ie sin.o g/jou nal/10.3389/ nana.2015.00037/
abs ac
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