Dis up ion o P o eoglycans in Neu al
Tube Fluid by ␤-D-XylosideAl e s
B ain Enla gemen in Chick Emb yos
M.I. ALONSO, A. GATO,*J.A. MORO, AND E. BARBOSA
Depa amen o deAna omı´a Humana, Facul ad de Medicina, 47005 Valladolid, Spain
ABSTRACT
Following neu ula ion, he an e io end o he neu al ube unde goes a
d ama icinc easeinsizeduemainly o heenla gingo heb ainca i y.This
ca i yis illedwi hso-calledneu al ube luid(NTF),whoseposi i ep essu ehas
been shown o play a key ole in b ain mo phogenesis. This luid con ains
a wa e -soluble ma ix, ich in chond oi in sul a e (CS), which has been
p oposedasanosmo ic egula o o NTF p essu e genesis. The pu pose o he
p esen s udy is o obse e he in luence o CS on NTF osmolali y and i s
ela ion oNTF hyd os a ic p essu e and b ain expansion.
NTFwas ob ained by means o mic oaspi a ion om he mesencephalic
ca i yo chickemb yos.Theosmolali yo NTFbe weenH.H.s ages20and29
wasmeasu edon he basis o i s c yoscopic poin .CSsyn hesis was dis up ed
by using ␤-D-xyloside and he induced a ia ions in b ain olume we e
measu edbymeanso mo phome y.Wealsomeasu ed he a ia ionsinNTF
osmolali y, hyd os a ic p essu e, and he concen a ion o CS and sodium
inducedbymeans o ␤-D-xyloside.
Ou da a e eal ha , a he ea lies s ages o de elopmen analyzed,
a ia ions in NTF osmolali y show a cha ac e is ic pa e n ha coincides
wi h hede elopmen al changes in he p e iously desc ibed luid p essu e.
Chick emb yos ea ed wi h ␤-D-xyloside, a chemical ha dis up s CS
syn hesis,displayedano ableinc easeinb ain olumebu noo he appa en
de elopmen al al e a ions. Mo phome ic analysis e ealed ha his inc ease
was due o hype enla gemen o he b ain ca i y. ␤-D-xyloside b ings abou
speci ic changes in he biochemical composi ion o NTF, which en ails a la ge
inc ease in CS concen a ion, mainly in he o m o ee chains, and in ha o
sodium. As a esul , he luid’s osmolali y and b ain in aluminal p essu e
inc eased,whichcouldaccoun o heinc easeinsizeo heb ainanlage.
These da a suppo he hypo hesis ha he in aluminal p essu e
in ol ed in emb yonic b ain enla gemen is di ec ly dependen on NTF
osmolali y, and ha he concen a ions o CS and i s associa ed mic oions
could play a key ole in he egula ion o his p ocess. Ana . Rec. 252:499–
508, 1998. 1998 Wiley-Liss, Inc.
Key wo ds: neu al ube; b ain de elopmen ; chond oi in sul a e; ␤-D-
xyloside; chick emb yo
The an e io end o he neu al ube, he u u e b ain
anlage,unde goesaconside ableinc easein olumea he
ea lies s ages o de elopmen , wi h a signi ican di e -
ence eme ging be ween he spinal co d and he u u e
encephalon. The mechanisms in ol ed in his impo an
mo phogene icp ocess emain la gely unknown o da e.
In he las decades, many e o s ha e been made in
o de o quan i y he a e o b ain enla gemen a hese
pa icula s ages o de elopmen . In chick emb yos, Pa-
G an sponso : Jun a de Cas illa y Leo´n and Ibe due o, Inc.;
G an sponso : Uni e sidad de Valladolid oA.Ga o.
*Co espondence o:D .AngelGa o,Depa amen odeAna omı´a
Humana, Facul ad de Medicina, C/ Ramo´n y Cajal 7, 47005 Val-
ladolid,Spain. E-mail: [email p o ec ed]
Recei ed13 June 1997;Accep ed6May 1998
THE ANATOMICAL RECORD 252:499–508 (1998)
1998 WILEY-LISS, INC.
checo e al. (1986) desc ibed an ini ial pe iod o mode a e
b ain g ow h om H.H. s ages 11-18 (Hambu ge and
Hamil on, 1951), ollowed by a pe iod o apid g ow h
be ween s ages 19 and 26. In his inal pe iod, and in only
48 h, he emb yonic b ain unde goes a 30- old size in-
c ease, ⬎70% o his being accoun ed o by g ow h o he
b ain ca i y (Desmond and Jacobson, 1977). The explosi e
g ow h o he emb yonic b ain ca i y has been di ec ly
ela ed o he expanding o ce gene a ed by he posi i e
p essu eo hein aluminal luiddesc ibedbyJelinekand
Pexiede (1968). Such p essu e equi es he p e ious
occlusion o he spinal co d lumen, ans o ming he b ain
ca i y in o a physiologically sealed sys em (Schoenwol
and Desmond, 1984; Desmond e al., 1993). The pa icula
ole, based on physical phenomena, ha hese mo phoge-
ne icmechanismscanplayin hede elopmen alpa e no
a ious emb yonic anlages was summa ized by Newman
and Compe (1990). In ac , expanding o ces based on
in aluminalhyd aulic mechanisms ha e been in ol ed in
he g ow h and de elopmen o o he anlages besides he
emb yonic b ain, such as he o ic esicle (Ba bosa e al.,
1986) and he eye (Coulomb e, 1956). An expe imen al
demons a ion ha he posi i e p essu e exe ed by NTF
con ined in he b ain ca i y is a key ac o in emb yonic
b ain expansion and mo phogenesis was p o ided by
Desmond and Jacobson (1977) and Desmond (1985). They
induced an expe imen al educ ion in his p essu e in
chick emb yos, which led o se e e dysmo phogenesis and
b aincollapse.
The composi ion and unc ion o he luid con ained in
he emb yonic b ain ca i y, and hei possible in luence on
ea lyb ainde elopmen , equi emo e esea ch(Jacobson,
1991). In a p e ious epo (Ga o e al., 1993), we sug-
ges ed ha he osmo ic p essu e o NTF could play a key
ole in he genesis o his expanding o ce and demon-
s a ed he p esence o a wa e -soluble ma ix ich in he
p o eoglycanchond oi insul a e (CS) in his luid.
Gi en he special osmo ic p ope ies o he p o eogly-
cans (PGs) (Compe and Lau en , 1978), we hypo hesized
ha he osmo ic p essu e o NTF could be, a leas in pa ,
egula edbyi s CS concen a ion.
Ino de ocla i y hemechanismsleading o hegenesis
o p essu eandi s egula ion, wemeasu ed heosmolali y
o NTF in chick emb yos om H.H. s ages 20 o 29. In
o de oanalyze he ela ionbe ween heCSconcen a ion
in NTF, he osmolali y o his luid and emb yonic b ain
expansion, we dis up ed he syn hesis o CS in he NTF
wi h ␤-D xyloside, a chemical ha selec i ely dis up s he
syn hesiso sul a ed PGs (Schwa z e al., 1976).
␤-D-xylosidesha e beenwidely employedin hes udyo
he oles played by sul a ed PGs (pa icula ly CS) a
di e en emb yonicde elopmen s agesin he a (Mo iss-
Kay and C u ch, 1982), chicken (Gibson e al., 1978,1979;
Kanke e al., 1982; Segen and Gibson, 1982), sea u chin
(Kinoshi a and Saiga, 1979), and og (Yos , 1990). ␤-D-
xylosides speci ically al e he syn hesis o hose sul a ed
PGs whose glycosila ion begins wi h he sequence xylosyl-
galac osyl-galac osyl,includingchond oi insul a eandi s
epime es de ma an sul a e, hepa an sul a e, and hepa in
(Schwa ze al., 1974; Robinson e al.,1975; Roden, 1980).
Howe e ,p-ni ophenyl-␤-D-xylopy anosideespeciallydis-
up schond oi inandde ma ansul a esyn hesis.Se e al
s udiesha eshowni sincapabili yo p iming hehepa an
sul a e and/o hepa in syn hesis in emb yonic issues
(Sobue e al., 1987; Lugemwa and Esko, 1991), o in issue
cul u es om di e en sou ces (Galligani e al., 1975;
Spoonce e al., 1983). The da a om he p esen s udy
indica e ha CS is he only sul a ed p o eoglycan in chick
emb yo NTF. The e o e, we can assume ha he changes
in NTF physicochemical p ope ies induced by means o
␤-D-xyloside a e undamen ally due o a dis up ion in CS
syn hesis.
MATERIALSAND METHODS
Fe ile Whi e Legho n eggs we e incuba ed a 38°C in a
humidi ied a mosphe e o ob ain chick emb yos a di -
e en de elopmen al s ages, anging om H.H. 20 o 29
(Hambu ge and Hamil on, 1951). A e incuba ion, a
small window was opened in he shell and he ea e he
emb yos we e classi ied acco ding o s age. Fo ␤-D-
xyloside ea men , doses o 24 µl o p-ni ophenyl-␤-D-
xylopy anoside4mM(Sigma,S .Louis,MO)we einjec ed
wi h a Hamil on mic osy inge in o he subge minal space
o H.H. s age 12–13 emb yos; he con ol g oup was
injec ed wi h s e ile saline solu ion. A e wa d, he open-
ing in he shell was sealed and he eggs we e eincuba ed
o 44h un il he emb yos eached H.H. s age 23.
Mo phome icAnalysis
The me hod used o de e mine b ain olume is e y
ime- consuming; he e o e, he numbe o emb yos o be
measu ed was limi ed o 10 con ol and 10 ␤-D-xyloside-
ea ed emb yos.A e ixa ion in Bouin’s solu ion o 6 h,
emb yos we e dehyd a a ed in g aded e hanol and embed-
ded in pa aplas ; 8 µm ans e se se ial sec ions we e cu
and s ained wi h haema oxylin-eosin. The olume was
calcula ed om he os al ex en o he b ain un il he
i s sec ion in which he o ic esicles appea ed, including
o eb ain, midb ain, and pa o he hindb ain. The inne
andou e limi s o heneu oepi helium we e d awninone
o e e y i e sec ions (sec ion ac o ⫽5) using a Lei z SM
Lux mic oscope equipped wi h a d awing ube ( o al
magni ica ion 60X), and he co esponding a eas in e-
g a ed wi h a Videoplan (Kon ol Elek onic GMBH) com-
pu e ized image-analysis sys em, ia he VIDAS 2.1.
s e eology p og am. In each emb yo, he olume o he
b ain, o i s ca i y, and ha o he neu oepi helial wall
we eob ainedbyadding he co espondingsec ional a eas
(in µm2) mul iplied by 8 µm ( hickness) and by 5 (sec ion
ac o ). Acco ding o Desmond and Jacobson (1977) and
Pacheco e al. (1986), he measu emen o e e y i h
sec ion di e s ⬍10% om he measu emen o e e y
sec ion in H.H. s age 23 chick emb yos, and his e o can
be ega dedasaccep able.The inal olumesa eexp essed
as he a i hme ic mean o he alues o he 10 emb yos ⫹/-
s anda d e o . Fo compa ison o he a e age olumes
( o al, ca i y, and issue) be ween expe imen al and con-
ol emb yos, wo- ailed S uden ’s - es o independen
samples was applied and P alues ⬍0.05 we e in e p e ed
asindica ings a is ical signi icance.
Elec on Mic oscopy
A e in o o ea men , he ex aemb yonic memb anes
we e excised and bo h con ol and ea ed emb yos insed
in saline solu ion. The p ima y b ain esicle egions we e
sec ioned om he emb yonic heads wi h hin ungs en
needles, unde dissec ing mic oscope con ol, in a Silga -
coa edPe idish.Theb ainsegmen swe e insedagainin
saline solu ion, ixed o e nigh in Ka no sky ixa i e
500 ALONSO ET AL.
con aining 2% annic acid a oom empe a u e, ollowing
he me hod o Singley and Solu sh (1980), and hen
pos ixed in 1% osmium e oxide o 1ha oom empe a-
u e. The samples we e dehyd a ed in a g aded ace one
se ies and embedded in plas ic (Spu , 1969). Ul a hin
sec ions om he di e en b ain esicles we e cu on an
AMC ul a-mic o ome, MT 6000 XL, s ained wi h u anyl
ace a e and lead ci a e (Reynolds, 1963) and examined
unde aJeol 1200 EX elec on mic oscope a 80 K .
Neu al Tube FluidAcquisi ion
NTF o osmolali yde e mina ionandbiochemicalanaly-
sis was ob ained as ollows. The ex aemb yonic mem-
b anes we e emo ed, he emb yos we e insed wice in
s e ile saline solu ion and placed in a d y Pe i dish, and
he su ounding luid was aspi a ed. A mic opipe e, con-
nec ed o a mic oaspi a o (Medical Sys em PLI 100), was
ca e ully placed in he middle o he mesencephalic ca i y
unde dissec ingmic oscope con ol, a oiding con ac wi h
he neu oepi helial wall. The NTF o se e al emb yos was
slowly aspi a ed, un il a su icien amoun was ob ained
o osmolali y de e mina ions (200 µl o each H.H. s age
be ween 20 and 29) and biochemical analysis (300 µl om
bo h con ol and ea ed H.H. s age 23 chick emb yos). NTF
wasaliquo edand ozen a -40°C un il i was o be used.
NTF Osmolali y De e mina ions
Measu emen s o NTF osmolali y we e made on he
basis o i s c yoscopic poin wi h an Osmoma 050 (Gono-
ec).Sincea minimum o 50 ul we e necessa y o accu a e
assessmen ,apool(200µl)o NTFwasmadewi h heNTF
ob ained om emb yos o each H.H. s age be ween 20 and
28,andano he pool(200µl) omcon oland␤-D-xyloside
ea ed H.H. s age 23 chick emb yos. Fou di e en de e -
mina ions o NTF osmolali y we e pe o med in all cases.
Mean and s anda d e o s we e calcula ed o each de el-
opmen al s age and he nonpa ame ic wo- ailed Mann-
Whi ney U es was used o analyse he di e ences in
osmolali y among s ages. P obabili y (P) alues ⬍0.05
we ein e p e edas indica ing s a is ical signi icance.
NTF P essu e De e mina ions
To measu e he in alumen p essu e o b ain anlage,
li e emb yos we e placed in a Pe i dish, as desc ibed in
he sec ion on NTF acquisi ion, and a mic opipe e o 30
µm (inne diame e ) connec ed o a mic oaspi a o was
ca e ully placed in he hombencephalic ca i y. Posi i e
p essu e o he lumen caused NTF o mo e in o he
mic opipe e. By o se ing his p essu e, using he ins u-
men balancing p essu e sys em, we es ima ed in alumi-
nal p essu e as ha p essu e necessa y o make he NTF
e u n o heb ainca i y.Onlymeasu emen s ha didno
show NTF leakage we e alued. A o al o 13 measu e-
men s o con ol emb yos and 14 o ea ed ones we e
made on H.H. 21 s age emb yos. Mean and s anda d
e o s we e calcula ed, and he wo- ailed S uden ’s es
o independen samples was applied in o de o compa e
he a e age p essu es o expe imen al emb yos wi h hose
o con ol emb yos, p obabili y (P) alues ⬍0.05 we e
in e p e edasindica ing s a is ical signi icance.
GlycosaminoglycansAnalysis
Isola ion and pu i ica ion o he NTF’s gly-
cosaminoglycans GAGs. The me hod o isola e and
pu i y he GAGs was based on ha p oposed by B een e
al. (1976). Aliquo s o NTF om con ol and ea ed
emb yos we e diges ed wi h p o ease ype XIV (Sigma) a
50°C o 6 h. The GAGs we e p ecipi a ed om he diges
by he addi ion o 5 olumes o 5% po assium ace a e in
absolu ee hanoland cen i uga ion a 17,500 g a 4°C.
Quan i a i eanalysiso GAGs.The o alamoun o
isola ed GAGs was es ima ed by he u onic acid-ca bachol
eac ion desc ibed by Bi e and Mui (1962). B ie ly, a e
igo ous hyd olysis in 0.025 M sodium e abo a e in
sul u ic acid, 200 µl o 0.125% ca bachol (Fluka) in abso-
lu e e hanol we e added. The op ical densi y was ead a
530 nm and he abso bance alues we e plo ed agains
hoseob ainedwi h s anda d solu ions o CS.
Quali a i e analysis o GAGs. The composi ion o
GAGsinNTFwasde e minedbymonodimensionalelec o-
pho esis and speci ic glycosidase diges ion. GAGs isola ed
om NTF o con ol and ea ed emb yos, as well as
s anda d solu ions o GAGs (Sigma), we e applied o
cellulose ace a e s ips (Cellogel 2.5 x 17), and elec opho-
esedin 0.1 M o mic acid-py ydinebu e ,pH 3.1, a 270
o 1 h acco ding o Beeley (1985). A e elec opho esis,
he s ips we e s ained wi h 1% alcian blue 8GX (Gu ) in
0.05 M ace a e bu e , pH 5.8, and e hanol ( / ). Fo
enzyma iciden i ica ion,glycosidaseschond oi inaseABC
and AC (Sigma) we e used, ollowing he diges ion p oce-
du e o B een e al. (1976). Chond oi inase ABC speci i-
cally diges s chond oi in 4- and 6-sul a e, and o some
ex en , hyalu onic acid, whe eas chond oi inase AC only
diges s chond oi in 4- and 6-sul a e (Yamaga a e al.,
1968). The ac i i y o he enzymes was p e iously checked
wi h s anda d GAGs. A e wa d, aliquo s o he GAGs
isola ed om NTF in con ol and ea ed emb yos we e
dilu ed in 50 µl o 0.15 M T is-HCL bu e con aining 0.5
IU o chond oi inaseABC (pH 8), o 0.5 IU o chond oi in-
ase AC (pH 7). The mix u es we e incuba ed a 37°C
o e nigh and gen ly shaken, and he GAGs we e isola ed
andelec opho esedas desc ibed below.
Sodium De e mina ion
Sodium concen a ion (bo h bound and ee) in NTF
om con ol and ea ed emb yos was measu ed by lame
emission spec oscopy on a Va ian Spec a A-80 wi h a
Va ian GTA-100 a achmen .
P o einAnalysis: Wes e n Blo
The o al concen a ion o p o eins in NTF was de e -
mined by he Bio-Rad p o ein assay based on he B ad o d
(1976) dye binding p ocedu e. Fo Wes e n blo analysis,
aliquo s o NTF om con ol and ea ed emb yos we e
mixed wi h a sample bu e con aining 3.2% SDS, 16%
glyce ol,2.8M␤-me cap oe hanoland0.0016%b omophe-
nol blue. Samples we e applied o a 4–20% g adien
SDS-PAGE gel (Bio-Rad) acco ding o Laemly (1970), and
he p o eins ob ained we e ans e ed o ni ocellulose
shee s, po e size 0.45µm(Bio-Rad), acco ding o he
me hod o Towbin e al. (1979). A e incuba ion o 2ha
oom empe a u e in he blocking solu ion (PBS con ain-
ing 5% skimmed milk), he ni ocellulose shee s we e
exposedo e nigh ,a 4°C, oan ichond oi insul a emono-
501CHONDROITIN SULFATE AND BRAIN ENLARGEMENT
clonal an ibody (CS-56 om Sigma), which ecognizes he
CS glycosidic ac ion (A uu and Geige , 1984), and
iden i ied wi h a pe oxidase-labeled mouse IgM PK 4010
Vec as ain a idin bio in complex ki (Vec o ). The pe oxi-
dase ac i i y was e ealed wi h diaminobenzidine (0.5
mg/mlinPBS wi h 0.02% hyd ogen pe oxide).
RESULTS
De elopmen al Pa e n o NTF Osmolali y
NTF osmolali y was measu ed in he pe iod o de elop-
men (H.H. s ages 20–29) in which apid b ain enla ge-
men occu s. Osmolali y de e mina ions we e no made in
p e ious s ages because o he impossibili y o ob aining
su icien amoun s o NTF.
NTF osmolali y a ia ions ollowed a cha ac e is ic pa -
e ndu ing heea lyde elopmen o chickemb yos(Fig.1).
NTF osmolali y oscilla ed a ound 250 mOsm/Kg wi h no
signi ican changes un il H.H. s age 24 (P⬎0.05, by
Mann-Whi ney U es ). Osmolali y hen d opped signi i-
can ly a s age 25 bu e u ned o 250 by s age 27. The e
we e s a is ically signi ican di e ences in alues o osmo-
lali ybe weens ages24and25andbe weens ages25and
26(P⫽0,029 by Mann-Whi ney U es ).
E ec o ␤-D-xyloside in B ain Enla gemen
A e in o o ea men , he b ain esicles exhibi ed
conside able g ow h wi h espec o he es o he neu al
ube and began o unde go seconda y esicula ion (Fig.
2-A). His ological obse a ion o hese emb yos (Fig. 2-C)
e eals ha he b ain anlage occupies mos o he emb y-
onicheadand e eals a la ge in e nal ca i y.
Emb yos ea edwi h␤-D-xylosideexhibi edmo phologi-
cal de elopmen simila o ha o he con ol specimens,
al hough hey all, o ag ea e o lesse deg ee, displayeda
signi ican inc ease in headsizewi h espec o hecon ol
specimens(Fig.2-B); hisseems obedue oahype enla ge-
men o he b ain esicles, whose mo phology was no
al e ed o any g ea ex en . No mo phological changes
we e seen in he de elopmen o o he emb yonic anlages
wi h he excep ion o he eye, which occasionally appea ed
smalle . The his ological sec ions o hese emb yos (Fig.
2-D)con i med he la ge inc ease inb ainanlagesize wi h
ega d o hecon olspecimens; hisseems obe he esul
o a global o e expansion o he b ain ca i y. These ␤-D-
xylosideinducedchangesa eunaccompaniedbyanyhis o-
logical al e a ions in he mo phology o he b ain anlage,
which shows no mal p og ess in b ain esicula ion; how-
e e ,some mo phologicalal e a ions canbeapp ecia eda
hebo de be ween hediencephalonand hemesencepha-
lon, such as a la ening in he diencephalo-mesencephalic
issu eandin he mesencephalic loo (Fig. 2C-D).
Mo phome ic da a (Fig. 3) indica e ha he size o he
b ain anlage o he ␤-D-xyloside- ea ed emb yos was 37%
la ge han ha o he con ol ones. B ain ca i y olume
inc eased 50% mo e han ha o he con ol specimens,
whe eas heinc easein heb ainneu oepi helialwallwas
only 11% in he ␤-D-xyloside- ea ed emb yos. S a is ical
analysis shows ha he o al and ca i y olumes o he
expe imen al g oups we e signi ican ly g ea e han o
he con ols ( P⬍0.001), whe eas di e ences in issue
olume we e no signi ican ( P⬎0.05). Such da a indica e
ha b ain olume inc ease induced by ␤-D-xyloside is
mainlydue o an o e expansion o he b ain ca i y.
Ul as uc u e o B ain Neu oblas s
Ou esul s indica e ha ␤-D-xyloside-induced dis up-
ion o CS biosyn hesis is accompanied by changes in he
ul as uc u al appea ance o he apical ends o b ain
neu oepi helial cells. In con ol emb yos, he apical end o
he neu oblas s we e joined by gap junc ions, wi h nume -
ous and conspicuous in e cellula spaces (Fig. 4A). The
apical cy oplasm o hese cells showed mi ochond ia and
in acellula memb ane o ma ions mo phologically com-
pa ible wi h endoplasmic e iculum, Golgi cis e ns and
la ge (200–300 µm diame e ) esicula o ma ions o a
mode a ely elec ondense con en . Nea he apical su ace
se e al small esicula o ma ions, which esemble he
cla h in-coa ed esicles in ol ed in endocy osis, could be
seen.
The mos ema kable ul as uc u al changes induced
by ␤-D-xyloside in neu oblas s (Fig. 4B) was a no able
inc ease in apical in acy oplasmic o ganelles, such as
Golgi complexes and, especially, in la ge esicula o ma-
ionsga he ing in he icini y o he nucleus and sca e ed
nea he apical su ace. The ul as uc u al appea ance o
hese esicles does no di e om hose desc ibed in
con olemb yos,excep in hei numbe and g ea e size.
␤-D-xyloside Induces Speci ic Changes in NTF
Biochemical Composi ion, Osmolali y,
and Hyd os a ic P essu e
Biochemical analysis o he NTF o con ol emb yos
(Table 1) e ealed ha , a H.H. s age 23, i s main compo-
nen is p o ein (295 ug/100ul),in which he PGs ep esen
a small pe cen age (12,5 ug/100 ul). In hese emb yos, he
esul s o he quali a i e analysis o he NTF (Fig.5)
iden i y wo di e en ypes o GAGs, CS and hyalu onic
acid. Howe e , he elec opho e ic sepa a ion o na i e
NTF (wi hou p io diges ion wi h p o ease) o emb yos o
he same s age e ealed no mig a ion o any o i s compo-
nen s, wi h he GAGs (iden i ied as posi i e alcian blue
ma e ial) emaining a he le el a which hey we e
Fig. 1. G aph showing he pa e n o NTF osmolali y a ea ly s ages o
chick emb yo b ain de elopmen . The osmolali y emains s able un il H.H.
s age 24; howe e , a ansi o y d op can be obse ed a H.H. s age 25.
The e olu ion o NTF osmolali y coincides wi h ha o b ain ca i y
hyd os a ic p essu e, which has been p e iously desc ibed. The alues
a e mean ⫹/- s anda d e o (n ⫽4). *
P
⫽0.0029 by Mann-Whi ney U
es .
502 ALONSO ET AL.
deposi ed (Fig. 6B2). This ac sugges s ha he GAGs
p esen in he NTF a e p obably in eg a ed in p o eogly-
canmac omolecula agg ega es,asoccu sino he ex acel-
lula ma ices. The esul s o he Wes e n blo , pe o med
o cla i y he na u e o NTF chond oi in sul a e, indica e
ha only a p o ein ac ion,M 197x103,wasp esen (Fig.7).
The esul s o o he de e mina ions on NTF composi-
ion, such as sodium concen a ion and osmolali y o he
con ol emb yos, we e o 2,316 ppm and 278 mOsm/Kg,
espec i ely(Table 1).
In he␤-D-xyloside ea edemb yos, hespeci icchanges
in he biochemical composi ion o NTF we e only e lec ed
in he PGs and ionic composi ion, wi h no al e a ion in i s
p o einconcen a ion(Table1).The u onicacidconcen a-
ion was 200% highe han ha o he con ol emb yos
(Table 1), and he esul s o he elec opho e ic sepa a ion
plus diges ion wi h speci ic glycosidases (Fig. 6A) indi-
ca ed ha his inc ease is exclusi ely due o CS, since he
amoun o hyalu onic acid emained unchanged. In he
elec opho esed na i e NTF o he ea ed emb yos (Fig.
6B), a band o simila mobili y o ha o CS appea ed,
which is highly sugges i e o he p esence o ee chains o
his p o eoglycan. Wes e n blo s o he NTF o he ea ed
emb yos wi h an ichond oi in sul a e CS-56 an ibody
showed (Fig. 7) ha CS elec opho e ic mobili y is simila
o ha o he con ol emb yos, wi h only a sligh educ ion
inepi opeconcen a ion.Theseda asugges ha ␤-D-xyloside
does no b ing abou subs an ial a ia ions in he molecula
weigh o he CS sec e ed in o he b ain ca i y. Ye ou
measu emen s o Na concen a ion o NTF o ␤-D-xyloside
emb yos compa ed wi h con ols showed nea ly a 40%
inc easein he o al sodium concen a ion (Table 1).
Ino de o cla i y he in luence o he inc ease o CS and
sodiumin heosmolali y o NTF,we measu ed hispa am-
e e inbo hcon oland␤-D-xyloside- ea edemb yos.The
esul s show ha ␤-D-xyloside ea men induces an 80%
inc ease in NTF osmolali y in compa ison wi h con ol
emb yos (Table 1). Fu he mo e, in o de o ind ou
whe he his inc ease in NTF osmolali y was ela ed o
changes in b ain in aluminal p essu e du ing he apid
b ain enla gemen pe iod, we measu ed he p essu e o
con ol and ea ed emb yos a s age 21 H.H. (a e 30 h o
␤-D-xyloside ea men ).As Table 2 shows, he in alumi-
nal p essu e in he b ain esicles o ea ed emb yos
inc eased 39% in compa ison wi h con ols, and his
di e ence is s a is ically signi ican (P⬍0.001).
DISCUSSION
In his epo , we es whe he he osmolali y o NTF is
di ec ly ela ed o hyd os a ic p essu e and whe he expe i-
Fig. 2. ␤-D-xyloside induces o e expansion o he emb yonic b ain
ca i y in chick emb yos. Al hough he mo phologic pa e n o b ain
esicula ion seems o be p ese ed, some al e a ions he diencephalo-
mesencephalic bo de s can be obse ed. Mac oscopic iew o H.H. s age
23 con ol (A) and ea ed (B) chick emb yos. Sagi al his ological sec ion
o con ol (C) and ea ed (D) emb yos. T, elencephalic esicles; D,
diencephalon; M, mesencephalon; R, hombencephalon. Scale ba s ⫽
1 mm.
503CHONDROITIN SULFATE AND BRAIN ENLARGEMENT

men ally induced changes in CS concen a ion o NTF by
meanso ␤-D-xylosidewouldlead osigni ican al e a ions
in NTF osmolali y and hyd os a ic p essu e, and how
muchchangesa ec b ain expansion in chickemb yos.
Onemajo inding, i.e., a signi ican d op in he osmolal-
i y o he NTF demons a ed by us, is consis en wi h he
d op in in aluminal p essu e epo ed by Jelinek and
Pexiede (1970). Howe e , we ound ha he changes in
hyd os a ic p essu e occu la e han he osmolali y
changes; his could be explained by he ime necessa y o
wa e in e change be ween NTF and emb yonic issues.
Fu he mo e, ou esul s show ha an expe imen ally
induced inc ease in he osmolali y o NTF by means o
␤-D-xylosideleads oasigni ican inc easeinin aluminal
p essu e o he b ain esicles and o a hype enla gemen
o heb ainanlageinchickemb yos.This ela ioncouldbe
explained i we bea in mind ha he osmo ic p essu e o
NTF implies an inc ease o wa e inside he b ain ca i y,
leading o an inc ease in hyd os a ic p essu e and/o a
neu oepi helial wall dis ension. This explana ion is based
on heassump ion ha heemb yonicb ainisaphysiologi-
cally sealed sys em (Schoenwol and Desmond, 1984;
Desmond e al., 1993). These da a suppo he hypo hesis
ha NTF osmolali y could be in ol ed in he con ol o
b ainexpansion.
Inaddi ion,we ha e al eady epo ed he p esence o an
osmo ically ac i e molecule, like CS, inside he b ain
ca i ies in chick emb yos (Ga o e al., 1993), and we ha e
also sugges ed ha he osmo ic p essu e o NTF could be,
a leas in pa , egula ed by i s CS concen a ion. Ou
s udy indica es ha NTF con ains a main p o ein compo-
nen , wo cha ac e ized PGs (CS and hyalu onic acid) and
high le els o sodium, all o which could con ibu e o NTF
osmolali y; howe e , he special osmo ic p ope ies o CS
(Compe and Lau en , 1978) make i a candida e o NTF
osmo ic p essu e egula ion. Mo eo e , ou esul s show
ha he changes in he CS concen a ion o NTF, expe i-
men ally induced by means o ␤-D-xyloside (wi hou any
o he associa ed change in he hyalu onic acid o he
p o ein componen o NTF), lead o signi ican modi ica-
ions in NTF osmolali y, hyd os a ic p essu e and b ain
enla gemen .
P o eoglycans ha e been p oposed as undamen al mol-
ecules in he egula ion o he wa e con en in biological
issues. Ce ain majo biological p ope ies o PGs a e he
esul o hei polyanionic na u e, which acili a es hei
in e ac ion wi h wa e in biological issues, hence hei
special osmo ic p ope ies and hyd aulic conduc i i y. CS
has he g ea es osmo ic capaci y,de e minedbyi su onic
acidcon en and hena u eo heglycosidicunion(Compe ,
1981; Compe and Zampa o, 1990), and ela i ely small
a ia ions in i s concen a ion p oduce la ge inc eases in
i sosmo iccapaci y (Compe and Lau en , 1978).
Ou esul s show ha he changes induced in CS
concen a iono NTFbymeanso ␤-D-xyloside,a eaccom-
panied by a signi ican inc ease in o al sodium concen a-
ion. ␤-D-xyloside is a chemical compound ha speci ically
dis up s sul a ed p o eoglycan biosyn hesis, bu we ha e
no ound any da a o jus i y a di ec in e e ence o his
compoundwi hionic anspo mechanisms; he e o e, he
changes in sodium concen a ion o NTF, induced using
␤-D-xyloside, seem o be ela ed o he CS a ia ions.
These da a sugges ha he e is a unc ional ela ion
be ween CS and sodium in NTF, and bo h molecules could
be co- esponsible o NTF osmolali y egula ion. Compe
and Lau en (1978) ha e shown ha sul a ed PGs bind
la ge amoun s o ino ganic ion (coun e ions) by elec o-
s a ic in e ac ions so ha he osmo ic p ope ies o PGs in
biological solu ions depend o a la ge ex en on he ac i y
o hei associa edcoun e ions.Ou da ashow ha sodium
isamajo ioniccomponen o chick emb yoNTF,soi sions
a e p obably he coun e ions associa ed wi h CS. The e-
o e, ou da a suppo he hypo hesis ha he NTF osmo-
lali y p ope ies in chick emb yos could be a ibu ed o a
coo dina ed egula ionin CS and sodium concen a ions.
Despi e he ac ha in aluminal PGs can egula e he
sodiumconcen a ion in NTFbyelec os a ic in e ac ions,
he e is a la ge sodium concen a ion in he NTF o chick
emb yos, which sugges s he exis ence o a sodium ans-
po mechanism ac oss he neu oepi helium owa d he
b ain ca i y. We do no ha e any e idence o suppo his
poin and u he esea ch is necessa y; howe e , Ba bosa
e al. (1985) ha e al eady epo ed he possibili y o
ansepi helial anspo o ions and wa e owa d he
inne ca i y o ano he ec ode mal de i ed anlage, he o ic
esicle. In addi ion, in he neu oepi helial cells o amphib-
ian emb yos, he p esence o sodium pumps, which ac-
i ely anspo sodium ou o he neu al ca i y, has been
desc ibedbyHo a yandRobinson (1990)and Bo gensand
Shi (1995). This ionic anspo gene a es a ansneu al
ubepo en ial ha seems obenecessa y o hes uc u al
in eg i y o he ea ly neu oepi helium and a p e equisi e
o no mal mo phogenesis. In amphibian emb yos, a
ansepi helial sodium anspo inside he emb yo has
beendesc ibedin heepiblas (Me cal andBo gens,1994),
and i seems o be p ese ed a e neu al ube in agina-
ion and in e naliza ion du ing neu ula ion o c ea e he
Fig. 3. Compa ison o b ain size o con ol and ␤-D-xyloside ea ed
H.H. s age 23 emb yos shows a signi ican dis up ion o he b ain
expansion p ocess. The o al b ain anlage olume o he ea ed emb yos
is 37% highe han ha o he con ol specimens. This inc ease is due o
b ain ca i y olume, which is 50% g ea e han ha o con ol specimens,
whe eas b ain issue mass inc eases by only 11%. The alues a e mean
⫹/⫺s anda d e o (n ⫽10).*
P
⬍0.001 by S uden ’s - es .
504 ALONSO ET AL.
Fig. 4. ␤-D-xyloside induces ul as uc u al changes in neu oepi helial
cells. Images a e om he apical pole o diencephalic neu oblas s o
con ol (A) and ea ed (B) H.H. s age 23 chick emb yos and show
changes induced by ␤-D-xyloside, in pa icula , a su p ising inc ease in
apical esicula o ma ions(V)andGolgi complexes (g).Scaleba ⫽1µm
in bo h igu es.
ansneu al ube po en ial. In he epiblas o a ian em-
b yos, a simila sodium anspo mechanism has been
desc ibed (S e n and MacKenzie, 1983); howe e , he
exis ence o a high NTF concen a ion o sodium a e
neu ula ion, as we ha e ound in ou wo k, sugges s ha
in chick emb yos he mechanism o sodium pumping om
he b ain ca i y has no been p ese ed, o pe haps has
been in e ed in o de o gene a e an osmo ic swelling
mechanism.
␤-D-xyloside induces ul as uc u al changes a he
apical end o b ain neu oblas s, mainly he inc ease in he
numbe o Golgi complexes and apical in acy oplasmic
esicles. Golgi complexes ha e been adi ionally associ-
a ed wi h glycosyla ion, undamen al o GAGs chains
syn hesis;simila ly, esicula o ma ions esembling hose
desc ibed ha e been ela ed wi h p o eoglycan syn hesis
and sec e ion ou o he cell (Hay, 1991). Mo eo e , he
p esence o nume ous esicules o simila appea ance and
con aining GAGs has been p e iously desc ibed in he
neu oblas s’ apical cy oplasm by Mak (1978) du ing he
usion p ocess o neu al olds in amphibian emb yos.
The e o e, i we bea in mind ha he ca i y o he b ain
TABLE 1. E ec s o ␤-D-Xyloside T ea men
on Neu al Tube Fluid Composi ion o H.H. S age 23
Chick Emb yos
T ea men P o eins
µg/100 µl
U onic
acid
µg/100 µl Sodium
ppm Osmolali y
mOsm/Kg
Con ol 295 12.5 2316 278
␤-D-xyloside 295 37.5 3259 504
Fig. 5. Two majo GAGs, hyalu onic acid and CS, we e iden i ied in
NTF o H.H. s age 23 chick emb yo b ains, by elec opho e ic sepa a ion
on cellulose ace a e combined wi h diges ion by speci ic glycosidases.
Lane 1: S anda d GAGs sepa a ion pa e n (HA: hyalu onic acid, KS:
ke a an sul a e, DS: de ma an sul a e, CS: chond oi in sul a e). Lane 2:
Elec opho e ic sepa a ion o GAGs ex ac ed om NTF o H.H. s age 23
chick emb yos. Lanes 3 and 4: GAGs ex ac ed om H.H. s age 23 chick
emb yo a e p e ea men wi h chond oi inase AC and ABC espec i ely.
The iden i ica ion o he wo bands as hyalu onic acid (a ow) and CS
(a owhead),was based on hei sensi i i y o chond oi inaseAC(Lane 3),
which speci ically diges s chond oi in-de ma an sul a e, and o chond oi i-
nase ABC (Lane 4), which diges s chond oi in-de ma an sul a e and
hyalu onic acid.
Fig. 6. Changes induced in NTF GAGs composi ion by means o
␤-D-xyloside alued by elec opho e ic sepa a ion on cellulose ace a e
combined wi h diges ion wi h speci ic glycosidases. A. Lane 1 shows he
elec opho e ic pa e n o s anda d GAGs (HA: hyalu onic acid, KS:
ke a an sul a e, DS: de ma an sul a e, CS: chond oi in sul a e). Lane 2:
Elec opho e ic sepa a ion o GAGs ex ac ed om NTF o ␤-D-xyloside
ea ed emb yos. Lane 3: The same as lane 2 a e chond oi inase AC
ea men . The elec opho e ic sepa a ion o GAGs ex ac ed om NTF
␤-D-xyloside ea ed emb yos shows ha mos GAGs mig a e o a simila
le elexhibi edby he chond oi inand de ma ansul a e s anda ds(Lane 2,
a owhead) and ha hey a e sensi i e o chond oi inase AC diges ion
(Lane 3). These ac s sugges ha he g ea inc ease in NTF u onic acid
concen a ion induced by means o ␤-D-xyloside is mainly due o
chond oi in / de ma an sul a e. B. The elec opho e ic pa e n o GAGs
om NTF in hei na i e s a e (wi hou p e ious diges ion wi h p o ease)
om␤-D-xyloside ea edemb yos,shows aband o simila mobili y oCS
(Lane 1, a ow), whe eas GAGs om con ol emb yos s ay a he same
le ela which hey we e deposi ed (Lane 2, a ow). These esul s sugges
ha in he NTF o con ol emb yos, he CS a e a anged o mac omolecu-
la agg ega es o PG, and ha ␤-D-xyloside p imes he syn hesis o CS
ee chains.
506 ALONSO ET AL.
esicles is delimi ed by he apical end o neu oblas s, we
can assume ha he CS p esen in he emb yonic b ain
ca i ycouldbeap oduc o apicalsec e iono heneu oepi-
helialcells.
Syn hesis and sec e ion o PGs a e maximally s imu-
la ed by he p esence o a ␤-D-xyloside, since his com-
pound a oids he physiological con ol mechanisms (Galli-
gani e al., 1975). The g ea inc ease in CS concen a ion
de ec ed in he NTF o chick emb yos ea ed wi h ␤-D-
xyloside sugges s a capaci y o PGs sec e ion a supe io
o ha occu ing in no mal condi ions, sugges ing ha i
mus beamechanism egula ing hesec e iono CS.I has
been sugges ed (Desmond, 1989) ha he expansi e p o-
cess o he chick emb yo b ain could be egula ed by he
sec e o y s a e o he neu oepi helium a each emb yonic
age. Ou esul s sugges ha his egula i e mechanism
could be based on he a e o CS sec e ion by he b ain
neu oblas s.
In conclusion, ou da a indica e ha NTF posi i e
p essu e, a majo mo phogene ic mechanism in ea ly
b ain de elopmen , migh be gene a ed by an osmo ic
wa e -binding phenomenon inside a physiologically sealed
b ainca i yand egula edbyconcen a ionso subs ances
ha a e osmo ically ac i e in NTF, among which CS and
i sassocia edmic oion, sodium, could play a key ole.
ACKNOWLEDGMENTS
We hank Sag a io Callejo, Ma ı´a La´za o, and Pila
Ma ı´n o echnical assis ance and D a. E. La o e o
cons uc i ec i icismo his manusc ip .
LITERATURE CITED
A uu Z,Geige B.Inmunocy ochemicallocaliza iono na i echond oi-
in-sul a e in issues and cul u ed cells using speci ic monoclonal
an ibody. Cell 1984;38:811–822.
Ba bosa E, Gi aldez F, Rep esa JJ T ansepi helial po en ial and
esis ance in he o ic esicle o he chick emb yo. J.Physiol. 1985;
365,53P.
Ba bosa E, Mo o JA, Ga o A, Rep esa JJ, Gi aldez F. P ecipi a e
ma he ial by CPC inside he o ic esicle o mouse and chick
emb yos.A ch Biol VII° Symp.In . Sciences Mo phologiques, 1986.
BeeleyJG.Glycop o einandP o eoglycanTechniques,Vol.16.Ams e -
dam:Else ie , 1985.
Bi e T, Mui HM.Amodi ied u onic acid ca bazole eac ion. Analy i-
calBiochem. 1962;4:330–334.
Bo gens R, Shi R. Uncoupling his ogenesis om mo phogenesis in he
e eb a e emb yo by collapse o he ube po en ial. De . Dyn.
1995;203:456–467.
B ad o d M. A apid and sensi i e me hod o he cuan i ica ion o
mic og am quan i ies o p o ein u ilizing he p inciple o p o ein-
dyebinding.Anal. Biochem.1976; 72:248.
B eenM,Weins einHG,BlachIJ,Bo che dingMS,Si igRAMe hods
Ca bohid . Chemi., 1976;7:101–115.
Compe WD. Polyme Monog aphs, Vol.7: Hepa in and Rela ed Poly-
saccha ides.NewYo k: Go don and B each, 1981.
Compe WD, Lau en TC. Physiological unc ion conec i e issue
polysacha ides.Physiol. Re . 1978;58:255–315.
Compe WD,Zampa o O. Hyd odynamic p ope ies o connec i e
issuepolysaccha ides. Biochem. J. 1990;269:561–564.
Coulomb eAJ. The ole o in aocula p essu e in he de elopmen o
he chick eye. I. Con ol o eye size. J. Exp. Zool. 1956;133:221–223.
Desmond ME. Reduced numbe o b ain cells in so-called neu al
o e g ow h.Ana . Rec. 1985;212:195–198.
DesmondME. P ecocious occlusion o he spinalco din chick emb yos
esul sinp ecociousexpansiono heb ain.Ana .Rec.1989;225:32–
33.
Desmond ME, Duzy MJ, Fede ici BD. Second messenge egula ion o
occlusion o he spinal neu ocoel in he chick emb yo. De . Dyn.
1993;197:291–306.
Desmond ME, Jacobson AG Emb yonic b ain enla gemen equi es
ce eb ospinal luid p essu e. De . Biol. 1977;57:188–198.
Galligani L, Hopwood J, Schwa z NB, Do man A. S imula ion o
syn hesis o ee chond oi in sul a e chains by ␤-D-xyloside in
cul u edcells. J. Biol. Chem. 1975;250:5400–5406.
Ga o A, Mo o JA, Alonso MI, Pas o JF, Rep esa JJ, Ba bosa E.
Chond oi in sul a e p o eoglycan and emb yonic b ain enla gemen
in he chick.Ana . Emb yol. 1993;188:101–106.
GibsonKD,Dolle HJ,Hoa RM.␤-D-xylosidescauseabno mali ieso
g ow h and de elopmen in chick emb yos. Na u e 1978;273:151–
157.
Gibson KD, Segen BJ, Dolle HJ Changes in chemical composi ion o
chick emb yos ea ed wi h a ␤-D-xyloside and La hy ogen. Te a ol-
ogy1979;19:345–356.
Hambu ge V, Hamil on HLA se ies o no mal s ages in he de elop-
men o he chick emb yo. J. Mo phol. 1951;88:49–82.
Hay ED.Cell Biolgy o Ex acellula Ma ix, 2nd ed. Bos on: Ha a d
MedicalSchool, 1991.
Ho a y KB, Robinson KR. The neu al ube o he Xenopus emb yo
main ains a po en ial di e ence ac oss i sel . De . B ain Res.
1990;59:65–73.
Jacobson M. De elopmen al Neu obiology, 3 d ed. New Yo k: Plenum
P ess,1991.
Jelinek R, Pexiede T. The p essu e on encephalic luid in chick
emb yosbe ween he2ndand6 hdayo incuba ion.Phisiol.Bhemo.
1961;97–305.
Fig. 7. Wes e n blo analysis o NTF o H.H. s age 23 con ol (CON)
and ␤-D- xyloside ea ed (BDX) chick emb yos. Monoclonal an ibody
CS-56 ecognizes a single band, M 197x10
3, in NTF o con ol emb yos.
In␤-D- xyloside ea ed emb yos, he ea eno appa en changes excep a
sligh educ ion in epi ope concen a ion.
TABLE 2. Resul s o he In aluminal P essu e, in
Con ol and ␤-D-Xyloside-T ea ed o H.H. S age 23
Chick Emb yonic B ainsa
Con ol (hPa) ␤-D-xyloside (hPa)
P essu e 37.8 ⫾1.5 (13)* 52.6 ⫾2.8 (14)*
aMeasu emen s a e mean ⫾s anda d e o . Numbe o em-
b yosmeasu ed a e inpa en heses.
*P⬍0.001,by wo- ailed S uden ’s es .
507CHONDROITIN SULFATE AND BRAIN ENLARGEMENT