Disruption o proteoglycans in neural tube fluid by B-

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. 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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