Basal lamina heparan sulphate proteoglycan is involved in otic placode invagination in chick embryos

Abs ac Fo ma ion o he o ocys om he o ic placode appea s o di e om in agina ion o o he cup-shaped o gan p imo dia. I is known ha he cellula cy oskele- on plays a limi ed ole in o ic placode in agina ion, whils he ex acellula ma ix unde lying he o ic p i- mo dium in e enes in he olding p ocess. In his s udy we ha e analysed he ole o he basal lamina hepa an sulpha e p o eoglycan in o ic p imo dium in agina ion. A 10 H.H. s age, hepa an sulpha e p o eoglycan im- munoma king begins o appea on he o ic placode basal lamina, inc easing no iceably a 13 H.H. s age, coincid- ing wi h maximum olding o he o ic epi helium, and is s ill p esen a la e s ages. Enzyme deg ada ion o hepa- an sulpha e p o eoglycan in he o ic p imo dium basal lamina, by means o mic oinjec ion wi h hepa inase III p io o olding, signi ican ly dis up s in agina ion o he o ic placode, which emains p ac ically la , wi h a sig- ni ican educ ion in he dep h o he o ic pi and an in- c ease in he diame e o he o ic opening. The immuno- cy ochemis y analysis e ealed a no able deple ion o basal lamina hepa an sulpha e p o eoglycan in he o ic p imo dia mic oinjec ed wi h hepa inase, wi h no s a is- ically signi ican di e ences obse ed in he olume o a e o cell p oli e a ion in he o ic epi helium ela i e o he con ol, which sugges s ha hepa an sulpha e p o - eoglycan dis up ion does no in e e e wi h he epi helial g ow h. In addi ion, a s udy o apop osis dis ibu ion by he TUNEL me hod con i med ha ea men wi h hepa- inase does no cause in e e ence wi h cell su i al in he o ic epi helium. Ou indings suppo he heo y ha o ic p imo dium in agina ion may be egula ed, a leas in pa , by he basal lamina componen s, which migh con ibu e owa ds ancho ing he o ic epi helium o adja- cen s uc u es. Key wo ds O ic de elopmen · Hepa inase · Mic oinjec ion · Epi helial olding · Ex acellula ma ix In oduc ion The o ic placode is an epi helial hickening de i ed om he su ace ec ode m and loca ed close o he hindb ain. In agina ion o o m he o ic pi coincides wi h he o - ma ion o se e al epi helial olds (Meie 1978; Hil e e al. 1989). In chick emb yos in agina ion s a s a 12 H.H. s age and ends a 16–17 H.H. s age wi h closu e o he o ic pi (Ál a ez and Na ascues 1990). The mechanisms con olling o ic placode in agina ion emain unclea . I has been pos ula ed ha he cellula cy oskele on plays a ole in he olding o nume ous emb yonal epi helial p i- mo dia (Hil e and Sea ls 1986), and changes in he o - ganisa ion o ac in and myosin could b ing abou con- s ic ion o cell apices, inducing epi helial in agina ion. In his ega d expe imen al manipula ion o ATP le els o dis up ion in he anspo o Ca++, condi ions ha in- e e e wi h he in e ac ion o cy oskele al ac in and myosin, ha e been shown o dis up in agina ion o he hy oid placode (Hil e e al. 1977), op ic esicle (B ady and Hil e 1982), nasal placode (Smu s 1981) and neu al pla e (Lee e al. 1983; Schoenwol e al. 1988; Fe ei a and Hil e 1993). Ne e heless, hese manipula ions ap- pa en ly ha e no e ec on in agina ion o he o ic pla- code (Hil e e al. 1989), which indica es ha he ac o s in ques ion do no play a decisi e ole in he epi helial olds leading o o ic esicle o ma ion. Ex acellula ac o s, o , mo e speci ically, di e se molecules o he ex acellula ma ix, ha e also been as- socia ed wi h epi helial in agina ion. In ac , i is known ha syn hesis dis up ion o enzyme deg ada ion o a ce - J.A. Mo o-Balbás · A. Ga o Ins i u o de Neu ociencias de Cas illa y León, Facul ad de Medicina, Valladolid, Spain e-mail: [email p o ec ed] M.I. Alonso · P. Ma ín · A. de la Mano Depa amen o de Ana omía, Facul ad de Medicina,Valladolid, Spain J.A. Mo o-Balbás (✉) Depa amen o de Ana omía Humana, Facul ad de Medicina, Uni e sidad de Valladolid, C/ Ramón y Cajal 7, 47005-Valladolid, Spain Ana Emb yol (2000) 202:333–343 © Sp inge -Ve lag 2000 ORIGINAL ARTICLE J.A. Mo o-Balbás · A. Ga o · M.I. Alonso · P. Ma ín A. de la Mano Basal lamina hepa an sulpha e p o eoglycan is in ol ed in o ic placode in agina ion in chick emb yos Accep ed: 2 June 2000 ain numbe o hese molecules such as collagen, ce ain p o eoglycans and glycop o eins may in e e e wi h epi- helial olding, as occu s in b anching o he sali a y glands, lung and kidney p imo dia (Klein e al. 1989; Nakanishi e al. 1986; Spoone and Faubion 1980; Spoone e al. 1985; Thompson and Spoone 1983), as well as in he in agina ion o he o ic p imo dium (Ge chman e al. 1991). I has been demons a ed ha ea men wi h uni- camycin, a d ug which inhibi s N-linked glycosyla ion, diminishing he syn hesis o glycosaminoglycans, dis- up s o ic p imo dium in agina ion (Yang and Hil e 1982; Rausch and Hil e 1988). Mo e ecen ly, Ge chman e al. (1995) ha e shown ha he des uc u ing o he ex acel- lula ma ix benea h he o ic p imo dium, by means o ea men wi h β-D-xyloside o by enzyme diges ion wi h es icula o s ep omyces hyalu onidase, signi ican ly dis up s o ic placode in agina ion due o al e a ion o he chond oi in sulpha e p o eoglycan in he ex acellula ma ix. The composi ion and s uc u ing o he basal lamina seems o play an impo an ole in o ic placode in agina- ion. The e is e idence ha a achmen o he o ic epi he- lium o he neu al ube is equi ed o no mal in agina- ion; he o ic placode became a ached o he neu al ec o- de m h ough a single laye o ib onec in and collagen IV be ween laye s o laminin (Hil e and Randolph 1993) and mic oinjec ion o an ibodies agains laminin de ach he wo p imo dia, in e e ing wi h in agina ion o he o ic placode (Ge chman e al. 1995). All his appea s o indica e ha o ic placode in agina ion depends o a la ge ex en on he composi ion and p ope s uc u ing o he ex acellula ma ix and basal lamina. O he p o eoglycans such as hepa an sulpha e a e p esen in he basal laminae o emb yonal p imo dia ha unde go in agina ion (Toole 1991), and could play a ole in epi helial olding, in his way, i is known ha expe i- men al dis up ion o hepa an sulpha e p o eoglycan (HSPG) al e s neu al old ele a ion du ing neu ula ion (Tuke and Mo iss-kay 1989) and inhibi s b anching mo phogenesis in emb yonic lung explan s (To iyama e al. 1997). Appa en ly HSPG is in ol ed in he in eg a- ion o he di e en componen s o basal lamina in p o- cesses ela ing o epi helial in e cellula adhesion and in es ablishing epi helium-mesenchyme ela ions (Jalkanen e al. 1991; Wigh e al. 1991; Paulsson 1992; Salmi i a and Jalkanen 1995). Since, as we men ioned abo e, HSPG is in ol ed in di e se ypes o epi helial olding du ing emb yonal de elopmen , we ha e ied in his s udy o de e mine whe he his p o eoglycan plays a ole in o ic placode in agina ion. We ha e de e mined ia im- muno luo escence he exp ession o HSPG in he basal lamina o he o ic placode be ween s ages 10 o 16 H.H., a pe iod comp ising placode in agina ion. We ha e also demons a ed i s in ol emen in he olding o he o ic epi helium, gi en ha he speci ic enzyme deg ada ion o his p o eoglycan dis up s o ic placode in agina ion. I is known ha HSPG can bind o se e al g ow h ac- o s, so his migh play a ole in egula ing he p oli e a- ion o cells (Jaakkola and Jalkanen 1999). In his ega d we ha e assessed epi helial olume, he inco po a ion a e o B dU and localisa ion o apop osis by he TUNEL me hod in he o ic epi helium. Ou indings indica e ha HSPG enzyme deg ading does no appea o in e e e wi h he su i al o o ic epi helial cells o o ha e an e ec on hei g ow h. Ma e ials and me hods Fo ou s udy we used Whi e Legho n chick e ile eggs (Ibe ec Fa ms, Valladolid), which we e incuba ed a 38°C in a o ced d a incuba o wi h 90% ela i e humidi y. HSPG-immunoma king In o de o s udy he HSPG exp ession we ob ained 10 o 16 H.H. (Hambu ge and Hamil on 1951) s age chick emb yos. Following ex ac ion he emb yos we e classi ied and ixed in Ca noy luid o 10 min and, a e apid dehyd a ion in e hanol, embedded in pa apla s o 1 h. The emb yos we e ans e sally o ien ed and se- ially sec ioned a 8 µm. Once ehyd a ed he sec ions we e washed in TRIS, pH 7.4 bu e , and we e ea ed wi h es icula hyalu onidase (15,000 U/ml in TRIS) o 30 min a 37°C. A e se e al inses in TRIS, he sec ions we e incuba ed in 1% TRIS/BSA o 20 min so as o block he possible union o he p i- ma y an ibody wi h non-speci ic p o eins. Incuba ion ollowed wi h he i s an ibody, Ra Ig-G an i-hepa an sulpha e p o eogly- can (Ups a e Bio echnology), in a solu ion o 10 µg/ml in 0.1% TRIS-BSA o 1 h a 37°C. A e se e al inses in TRIS bu e , he sec ions we e incuba ed in he seconda y an ibody, a 1/320 dilu- ion o an i- a Ig-G FITC conjuga e (Sigma), o 1 h a 37°C. Fol- lowing se e al inses wi h TRIS bu e , he sec ions we e moun ed in an aqueous medium (Aquamoun ). Fo isualisa ion and pho o- g aphing o he p epa a ions we used a con ocal mic oscope (Zeiss LSM-310). Con ol sec ions we e p epa ed as desc ibed abo e bu using p e-immune se um as he p ima y an ibody. Hepa inase mic oinjec ion Mic oinjec ing was ca ied ou “in o o”, subsequen o he open- ing o a small window in he egg shell. A China ink Ringe solu- ion was subge minally injec ed in o de o imp o e isualisa ion o he emb yo, which was hen classi ied by age. Only 9 and 10 H.H. s ages emb yos we e mic oinjec ed. Once he i elline memb ane had been cu wi h ungs en needles, subec ode mal mic oinjec ion was unde aken, wi h 2 nl o a solu ion o 4 U hepa inase III om Fla obac e ium hepa inum (Sigma) dissol ed in 5 µl Ringe , on he igh side o he emb yo la e al o he hindb ain. On he le side, used as he con ol, he same amoun o hea -deac i a ed en- zyme was mic oinjec ed. Mic oinjec ion was e ec ed wi h 10-µm- ip diame e mic oneedles connec ed o a mic oinjec o (Medical Sys ems, G een ale, N.Y. 11548, PLI-100). When mic oinjec ion had been comple ed, he egg shell was sealed wi h a plas ic adhe- si e ape and incuba e esumed o a pe iod o 9 h; hen, mic oin- jec ion in he same condi ions was epea ed, and once again e-in- cuba ion ook place o a u he 9 h, un il he emb yos eached s age 15–16 H.H. A o al o 112 emb yos we e mic oinjec ed and, in all o hem, he e ec p oduced by ea men wi h hepa inase was checked by di ec isualisa ion and compa ison wi h he ea ed side ( igh ) ela i e o he con ol side (le ), and he wid h o he o ic pi opening was no ed. The mic oinjec ed emb yos we e di ided in di e en se ies o subsequen p ocessing: 1. HSPG-immunoma king, ollowing he same me hod as he one desc ibed abo e 2. Ligh mic oscopy and mo phome ic analysis 334 3. Apop osis s udy by he TUNEL me hod 4. S udy o cell p oli e a ion by B dU inco po a ion 5. Scanning elec on mic oscopy Ligh mic oscopy and mo phome ic analysis A e mic oinjec ion he emb yos we e ex ac ed om he egg, insed in a 37°C Ringe solu ion and he amnio ic memb ane e- mo ed; his comple ed, hey we e ixed o a pe iod o 6 h Bouin's luid a oom empe a u e. Following dehyd a ion by imme sion in g aded e hanol, he emb yos we e embedded in pa aplas and 8-µm- ans e sal sec ions we e ob ained; hese we e p ocessed o s aining wi h haema oxylin-eosin. The mos ep esen a i e sec ions we e pho og aphed wi h a Nikon mic opho -FXA pho omac oscope. In he his ological sec ions we made h ee ypes o measu e- men s o assess he deg ee o g ow h and epi helial in agina ion: 1. Calcula ion o o ic epi helium olume 2. Measu emen o o ic pi dep h 3. Measu emen o he wid h o he o ic pi opening In o al, 8 emb yos we e used in hese ope a ions In o de o calcula e he epi helial olume o he o ic p imo - dia, he inne and ou e limi s o he o ic epi helium we e d awn in all sec ions om 16 H.H. s age emb yos, by means o a Lei z SM Lux mic oscope equipped wi h a d awing ube ( o al magni ica ion ×250). The epi helial a eas we e measu ed wi h a Videoplan (Kon- ol Elek onic) compu e ised image analysis sys em, wi h he VIDAS 2.1 s e ology p og am applica ion. Wi h a iew o ob ain- ing he o al olume o each o ic p imo dium, he a eas (exp essed in µm2) we e added and mul iplied by 8 µm ( he hickness o he sec ions). The inal olumes we e exp essed as he a i hme ic a e age o he alues ob ained±s anda d e o . The diame e o he o ic pi opening was measu ed c anio- caudally. F om he alues ob ained he a i hme ic mean was calcu- la ed and he inal esul s we e exp essed in µm±s anda d e o . The dep h o he o ic pi was always measu ed in he same a ea, in he middle o he o ic p imo dium; a his le el we aced a angen o he edges o he o ic pi opening, hen ano he line pe pendicula o he p e ious one as a as he deepes a ea o he o ic epi helium (Fig. 1). We conside ed his dis ance he maxi- mum dep h o he o ic pi . F om he alues ob ained he a i hme- ic mean was calcula ed and he inal esul s exp essed in µm±s anda d e o . To assess he possible di e ences be ween he o ic p imo dia ea ed wi h hepa inase ela i e o he con ols, in all measu e- men s a wo- ailed S uden - es o independen samples was ap- plied and P alues below 0.01 we e in e p e ed as indica ing s a- is ical signi icance. TUNEL me hod We used his echnique o de e mine he possible e ec s o he enzyme-deg ading o hepa an sulpha e p o eoglycan on he su i al o o ic epi helium cells whils assessing apop osis. The mic oinjec ed emb yos we e ixed in 4% o maldehyde-PBS and we e embedded in pa aplas and la e sec ioned a 8 µm. To localise apop osis we employed an Apop osis De ec ion Sys em, Fluo escein Ki (P omega) acco ding o he manu ac u e ’s ecom- menda ions. This sys em measu es he agmen ed DNA o apop o ic cells by ca aly ically inco po a ing luo escein-12-dUTP a he 3'-OH DNA ends using he enzyme e minal deoxynucleo- idyl ans e ase, which o ms a polyme ic ail using he p inciple o he TUNEL (Td -media ed dUTP Nick-End-Labeling) assay. Fo isualisa ion and pho og aphing o he p epa a ions we used a con ocal mic oscope (ZEISS LSM-310). Cell p oli e a ion assay In o de o de e mine whe he HSPG enzyme-deg ading in e - e es wi h cell p oli e a ion in he o ic epi helium, we made a s udy o B dU inco po a ion. Following ea men wi h hepa in- ase III and subsequen e-incuba ion o he emb yos, we admin- is e ed 20 nl o a 3% solu ion o B dU (Sigma) in PBS by in a- ca diac mic oinjec ion. One h la e he emb yos we e ixed in Ca noy and embedded in pa aplas . Eigh -µm sec ions we e de- pa a inized, ehyd a ed in descending se ies o e hanol, and he endogenous pe oxidase blocked by ea ing he sec ions wi h 0.5% hyd ogen pe oxide in me hanol o 30 min. A e se e al inses in PBS, he sec ions we e incuba ed in 4 M HCl a 37°C o 15 min. HCl was neu alized by se e al washes wi h 0.1 M sodium bo a e bu e , pH 8.5. A e being insed wi h PBS, he sec ions we e incuba ed wi h 6 µg/ml o an i-B dU an ibody (Dako) dilu ed in PBS a 4°C o e nigh . De ec ion o his an i- body was ca ied ou by means an An imouse Ki Ex a idin Pe oxidase (Sigma) acco ding o he manu ac u e 's ins uc ions. A sligh coun e colou ing wi h Ha is Haema oxylin was pe - o med. In o de o quan i y he deg ee o B dU inco po a ion in he o ic epi helium cells we coun ed he numbe o s ained nuclei in he o ic pi in al e na i e sec ions o e a su ace o 2,885 µm2in each cu ; his su ace was andom in he di e en sec ions. We used a o al o 6 emb yos and he esul s we e exp essed as he mean o s ained nuclei pe su ace±s anda d e o . To assess whe he di e ences exis in cell p oli e a ion be ween o ic p i- mo dia ea ed wi h hepa inase ela i e o he con ols a wo- ailed S uden's - es o independen samples was applied and P alues below 0.01 we e in e p e ed as indica ing s a is ical sig- ni icance. Scanning elec on mic oscopy Upon ex ac ion he emb yos unde wen ca e ul insing in a 37°C Ringe solu ion and he amnio ic memb ane was emo ed. Fol- lowing ixing in 2.5% glu a aldehyde in 0.1 cacodyla e bu e , he emb yos we e dehyd a ed in ace one. Subsequen ly, he emb yos unde wen c i ical poin d ying wi h liquid CO2in a Balze 's CPD de ice. Once he emb yos had been d ied, hey we e moun ed on a me al pedes al and co e ed wi h gold ilm wi h a Balze 's SCD- 310 sys em. A Jeol T-300 scanning elec on mic oscope was used o isualising and pho og aphing he samples. 335 Fig. 1A,B Schema ic d awing o show how we measu ed he dep h o he o ic pi . Fi s o all we aced a angen o he edges o he o ic pi opening and hen ano he line pe pendicula o his up o he deepes a ea o he o ic epi helium; we conside ed his dis- ance (d) he maximum dep h o he o ic pi . ALe o ic placode (con ol). BRigh o ic placode ( ea ed wi h hepa inase III) 336 Fig. 2A–G Mic og aphs a o ic p imo dium le el o chick emb yos, p ocessed wi h an i-hepa an sulpha e p o eoglycan (HSPG) an ibody and using a conjuga e seconda y an ibody wi h luo escein iso hiocyana e (FITC). AA s age 10 H.H., he HSPG o ms small pa ches a basal laminae le el in he o ic placode and hindb ain. BA s age 11 H.H., he HSPG o ms a con inuous laye o low luo escence, benea h he o ic epi- helium. CS age 12 H.H. The immunoma king agains HSPG shows a simila pa e n o ha o he p e ious s age; obse e he close con ac be ween he basal laminae o he o ic epi helium and he hindb ain neu oepi helium (a ows). D,E: S age 13 and 14 H.H. emb yos, espec i ely: he e we can obse e he no able inc ease, wi h espec o he p e ious s ages, in luo escen in ensi y a o ic p imo dium basal lamina le el, coincid- ing wi h maximum olding o he o ic epi helium. A s ages 15 H.H. (F) and 16 H.H. (G), HSPG-immunoma king o he o ic epi helium basal lamina is simila o he s age 14 H.H bu he egion nea he opening o he o ic pi shows li le ma king a s age 15 H.H., as also occu s a s age 16 H.H. in he la e al pa o he o ocys , when he o ic opening has now closed. All images a e o he same magni ica ion. Ba 50 µm s age 12 H.H. A his s age he basal lamina o he hind- b ain neu oepi helium shows an immunoma king pa - e n simila o ha o he o ic p imo dium; i s do so-la - e al po ion is in close con ac wi h he medial pa o he o ic placode and bo h basal laminae a e a ached (Fig. 2C). Du ing 13 and 14 s ages (Fig. 2D,E) a g ea e change is seen in he shape o he o ic placode as a esul o he olding o he epi helial wall, which leads o i s sinking in o he unde lying mesenchyme. This epi helial olding is mo e accen ua ed in he la e al po ion o he o ic pla- code, whils he medial pa unde goes ha dly any old- ing and becomes loca ed e y close o he do so-la e al wall o he hindb ain. A hese s ages and in coincidence wi h o ic p imo dium in agina ion he e is a signi ican inc ease, in espec o p e ious s ages, in HSPG-im- munoma king in ensi y on he basal lamina o he o ic pi . The medial pa o he o ic placode is s ill e y close o he hindb ain neu oepi helium, bu HSPG-immuno- ma king e eals ha i s basal laminae a e independen , in con as wi h s age 12 H.H. Resul s HSPG exp ession du ing o ic placode in agina ion We ha e s udied he empo al e olu ion o o ic placode HSPG exp ession by indi ec immuno luo escence be- ween s ages 10 and 16 H.H., a pe iod du ing which in- agina ion o he o ic p imo dium akes place, wi h he subsequen o ma ion o he o ic esicle. A 10 H.H. s age he o ic epi helium, si ua ed la e ally o he hindb ain, begins o hicken, and like he es o he su ace ec ode m, shows ha dly any HPGS-immuno- ma king on he basal lamina; only a ew discon inuous pa ches o low luo escence can be seen. Sligh signs o immunoma king a e also isible on he basal lamina o he hindb ain neu oepi helium (Fig. 2A). A 11 and 12 H.H. s ages (Fig. 2B,C), his hickening o he o ic epi helium is mo e p onounced han du ing he p e ious s age, bu he e a e s ill no e iden signs o in agina ion. On he o ic placode basal lamina a con in- uous HSPG laye appea s, becoming mo e luo escen a A s age 15 H.H. o ic epi helium in agina ion con in- ues, p incipally a he expense o olding o he mos me- dial po ion. The edges o he o ic pi come close o- ge he bu he e is s ill no con ac be ween hem, whils he o ic p imo dium ca i y emains in communica ion wi h he amnio ic ca i y ia a small po e (Fig. 2F). The HSPG-immunoma king pa e n is simila o ha o p e- ious s ages (13 and 14 H.H.), al hough he e is less in- ense luo escence in he o ic epi helium basal lamina in egions close o he opening o he o ic pi (Fig. 2F). A s age 16 H.H. he edges o he o ic pi ha e es ab- lished con ac and used o o m he o ic esicle, al hough he la e is s ill joined o he su ace ec ode m (Fig. 2G). The an ibody an i-HSPG ma ks a con inuous laye on he basal lamina o he o ocys wi h an in ensi y o luo es- cence simila o ha o he p e ious s age (15 H.H.), wi h he excep ion o he la e al pa o he o ic epi helium, which s ays joined o he ec ode m (Fig. 2G). The e ec o ea men wi h hepa inase III on o ic placode in agina ion In o de o de e mine whe he he HSPG p esen in he o ic placode basal lamina plays a ole in i s in agina ion, we ca ied ou enzyma ic deg ada ion by mic oinjec ing hepa inase III in o he mesenchyme unde lying he igh o ic p imo dium a s ages 9 o 10 H.H. chick emb yos. The hea -inac i a ed enzyme was mic oinjec ed in he le o ic p imo dium, which was used as he con ol. Supe icially, in almos all emb yos he o ic pi open- ing was wide on he side mic oinjec ed wi h hepa inase III ( igh ) han on he con ol side (le ), and in he mo e de eloped emb yos (s age 16 H.H.), he o ic opening had closed on his side. In emb yos p ocessed o scanning elec on mic osco- py (Fig. 3), i can be obse ed how on he igh side, mi- c oinjec ed wi h hepa inase III, he o ic p imo dium has unde gone ha dly any in agina ion and emains p ac i- cally la wi h a la ge opening o communica ion wi h he ex e io , as opposed o he le (con ol) side, in which in agina ion has been no mal and he o ic placode has sunk, despi e he e being a small opening communi- ca ing wi h he amnio ic ca i y. The emainde o he emb yo appea s mo phologically no mal on bo h sides. This shows ha al e a ions induced by ea men wi h hepa inase a e es ic ed o he icini y o he mic o- injec ed a ea. Ligh mic oscopy images (Fig. 5) e eal he same de- ec wi h ega d o in agina ion o he igh o ic placode ( ea ed wi h hepa inase III), and olding o he whole o he medial hal is seen o be non-exis en ; in his a ea he o ic epi helium occasionally appea s o ha e unde gone a sligh de-s uc u ing, losing he columna o ien a ion o i s nuclei (Fig. 5C), wi h espec o he le (con ol) o ic anlage (Fig. 5A), which appea s pseudo-s a i ied. Fu - he mo e, he hindb ain neu oepi helium and he medial po ion o he o ic epi helium a e mo e sepa a ed on he side mic oinjec ed wi h hepa inase III ( igh ) han on he con ol side (le ; Fig. 5A,C). F om a his ological poin o iew, he la e al po ion on he igh side ( he one ea ed wi h hepa inase III) o he o ic epi helium is no - mal, simila o ha o he con ol side (le ), wi h he cell 337 Fig. 3. A Scanning elec on mic og aphs showing he do sola e al su ace o he cephalic ex emi y o a s age 15 H.H. chick emb yo. The igh o ic placode ( OP) has been ea ed wi h hepa inase III; obse e he small deg ee o in agina ion wi h espec o he le , con ol o ic placode (lOP), ea ed wi h he same hea de-ac i a ed enzyme. Ba 200 µm. B,C Ampli ica ion o he image shown in A, co esponding o he igh and le o ic p imo dia, espec i ely. Ba 25 µm nuclei on bo h sides a anged in a cha ac e is ically pseudo-s a i ied s uc u e (Fig. 5A,C). These indings indica e ha enzyma ic deg ada ion o basal lamina HSPG a ec s he whole p ocess o o ic placode in agina ion, al hough his in luence appea s o be mo e e iden in he medial egion o he o ic pi (nex o he hindb ain neu o- epi helium). Al e a ions in o ic placode in agina ion b ough abou by ea men wi h hepa inase III a e accompanied by acu e changes in he HSPG dis ibu ion pa e n in he basal lamina. Figu e 4 shows he e ec i eness o enzyma ic diges ion a e 18 h o ea men . The igh o ic placode ( ea ed wi h hepa inase III) has no unde - gone in agina ion and a no able educ ion can be ob- se ed in HSPG-immunoma king on i s basal lamina (Fig. 4C) wi h espec o he le (con ol) o ic p imo - dium, which has now closed so as o o m he o ic esi- cle. This side has an in ense posi i e luo escen HSPG-band which aces he basal lamina o he o o- cys (Fig. 3A), excep in he closu e egion o he o ic pi , which emains in con ac wi h he su ace ec o- de m, his immunoma king pa e n co esponds o he one al eady men ioned o he no mal o ic p imo dium in 16 H.H. s age emb yos. 338 Fig. 4A–C HSPG-immunoma king o a ans e sal sec ion o a s age 16 H.H. emb yo a o ic p imo dium le el, showing he e ec- i eness o enzyme ea men . BThe igh o ic placode ( OP), mi- c oinjec ed wi h hepa inase III has no unde gone in agina ion, and i s basal lamina shows li le immuno luo escence compa ed wi h he con ol side (lOP), o which in agina ion has been no - mal. A,C Ampli ica ion o he igh and le o ic p imo dia p e- sen ed in B. Ba s B100 µm; A,C 50 µm Fig. 5A–C T ans e sal sec ion o a s age 15 H.H. emb yo a o ic p imo dium le el. B We can easily obse e a la ening wi h abso- lu ely no in agina ion a he igh o ic p imo dium ( OP) le el, as compa ed wi h he le , con ol o ic p imo dium (lOP), which has unde gone no mal in agina ion and e ains a small po e in com- munica ion wi h he amnio ic ca i y. A,C ampli ica ion o he igh and le o ic p imo dia shown in B. A sligh de-s uc u ing o he epi helium in i s medial po ion (a ow) can be obse ed in igh o ic placode (C). In he con ol side (A) he o ic epi helium appea s mo phologically no mal, wi h i s cha ac e is ic pseudo- s a i ied s uc u e. Ba s B100 µm; A,C 50 µm Mo phome ic analysis In o de o de e mine whe he ea men wi h hepa inase a ec s o ic epi helium g ow h and so as o e alua e he ex en o in e e ence in o ic placode in agina ion in- duced by deg ading he HSPG in he basal lamina, we made h ee di e en measu emen s in he his ological sec ions: (1) Calcula ion o he olume o he o ic epi he- lium, (2) Measu emen o he dep h o he o ic pi , (3) Measu emen o he diame e o he o ic pi opening. In all he measu ings, and in o de o compa e he ea ed side wi h ha o he con ol, a wo- ailed S u- den 's - es o independen samples was applied and P alues below 0.01 we e in e p e ed as indica ing s a is i- cal signi icance. The alues ob ained a e summa ised in Table 1. Compa ison o he epi helial olumes o he o ic p i- mo dia ea ed wi h hepa inase III and he con ols e- ealed no s a is ically signi ican a ia ions (P>0.01). This sugges s ha deg ading he basal lamina HSPG does no in e e e wi h o ic epi helium g ow h. Howe e , he dep h o he o ic pi was mo e han double on he con ol side, whils he diame e o he o ic pi opening was conside ably la ge ( ou imes) on he side ea ed wi h hepa inase III. Bo h measu emen s e lec ed s a is i- cally signi ican di e ences (P<0.01) in he deg ee o in- agina ion be ween he igh o ic pi ( ea ed side) and he le (con ol side). This shows ha enzyme deg ading o he basal lamina HSPG clea ly in e e es wi h o ic placode in agina ion. S udy o apop osis and cell p oli e a ion In o de o check whe he enzyme deg ading o basal lamina HSPG in e e es wi h cell su i al in he o ic epi- helium, we analysed a his le el he p esence o apop- osis by he TUNEL me hod. On he con ol side (le ) apop osis was only obse ed on he edges o he o ic opening, and coincided wi h he closu e and usion and subsequen isola ion o he o ic pi om he su ace ec o- de m in he o ma ion o he o ic esicle (Fig. 6C). As was o be expec ed, on he side ea ed wi h hepa inase III ( igh ) he e was a conside able delay in o ic pi in- agina ion and only one a ea o apop osis could be ob- se ed on he la e al edge o he o ic pi opening, in he ansi ion zone be ween he o ic epi helium and he ce- 339 Table 1 Compa ison o epi helial olume, o ic pi dep h, diame e o he o ic pi opening and cell p oli e a ion, be ween o ic p imo dia ea ed wi h hepa inase III ( igh o ic anlage) and he con ols (le o ic anlage). Values a e gi en as mean±s anda d e o . A wo- ailed S uden 's - es o independen samples was applied and P alues below 0.01 we e in e p e ed as indica ing s a is ical signi icance Epi helial olume Dep h o he o ic pi Opening o he o ic B dU-labeled (µm3×103) (µm) pi (µm) cells/a eaa Righ o ic anlage 1730.12±97.04 40.93±10.09* 237.33±34.36* 25.3±6 Le o ic anlage 1720.83±99.69 87.66±12.22* 56±36.48* 27.7±2.45 (n=8) (n=8) (n=8) (n=6) * P<0.01 aA ea=2885 µm2 Fig. 6 B dU immunolabelling (A,B) and apop osis de ec ed by he TUNEL me hod (C,D) in s age 16 H.H. chick emb yos. AO ic esicle o he le side (con ol). BRigh o ic placode ea ed wi h hepa inase III, showing a clea delay in in agina ion ela i e o he le side. On bo h sides s aining in ensi y and he numbe o nuclei s ained wi h B dU is simila , and cells in he DNA syn hesis phase a e loca ed mainly a he basal po ion o he o ic epi helium. Apop osis on he con ol side (C) was only obse ed in he egion o closu e and usion o he o ic opening (a ows). On he side ea ed wi h hepa inase III (D) he o ic placode has ba ely unde - gone in agina ion and apop osis is p esen solely on he la e al edge o he o ic pi (a ows). Occasionally we ound isola ed apop osis in he hindb ain neu oepi helium (a owheads) on bo h sides nea o he o ic pi . Ba s 50 µm phalic ec ode m (Fig. 6D). Apa om in hese a eas, which can be conside ed physiological o ones in which cellula dea h is no mal, we ound no sign o apop osis in he es o he o ic epi helium. We also encoun e ed se e al isola ed cell dea hs in he do sola e al wall o he hindb ain neu oepi helium on bo h he con ol side and ha ea ed wi h hepa inase III (Fig. 6C,D). By s udying B dU inco po a ion, we ound ha mos cells in he DNA syn hesis phase we e loca ed nea o he basal po ion o he o ic epi helium and ha he e we e ew cells s ained wi h B dU in he icini y o he apical po ion on bo h he side ea ed wi h hepa inase III and he con ol side (Fig. 6A,B); wha is mo e, s aining in ensi y on bo h sides was simila . In o de o check whe he HSPG enzyme deg ading in e e es wi h he p oli e a ion o cells in he o ic epi helium, we coun ed he cells s ained wi h B dU pe su ace uni (Table 1). We obse ed ha he e was a sligh dec ease in B dU in- co po a ion on he side ea ed wi h hepa inase ela i e o he con ol side; howe e , he s a is ical s udy e- ealed no s a is ically signi ican di e ences be ween he wo sides (P>0.01), which sugges s ha he dis up ion o basal lamina HSPG does no in e e e wi h cell p oli e a- ion in he o ic epi helium. Discussion The basal lamina o he o ic placode du ing in agina ion has a complex s uc u e, and he p esence o di e en componen s such as laminin, ib onec in, collagen IV and cy o ac in has been desc ibed (Richa dson e al. 1987; Hil e and Randolph 1993). Bo h quali a i e and quan i a i e di e ences exis in he dis ibu ion o hese molecules in he o ic placode. Cy o ac in ends o be lo- ca ed in he la e al po ion, whils he medial pa o he o ic p imo dium comes in o con ac wi h he neu al ube neu oepi helium; a his le el he basal laminae o bo h epi helia a e a ached by hin laye s o laminin, collagen IV and ib onec in (Hil e and Randolph 1993) and he e is da a ha indica es ha a achmen o he o ic epi- helium o he neu al ube is equi ed o no mal in agi- na ion. In his ega d i has been demons a ed ha mic oinjec ion o an ibodies agains laminin de ach he wo p imo dia, in e e ing wi h o ic placode in agina ion (Ge chman e al. 1995). In ou s udy we ha e shown ha he HSPG o ms pa o he o ic placode basal lamina, and we ha e demons a ed ha he e is a p og essi e inc ease in he concen a ion o his p o eoglycan om s ages 10 o 13 H.H., when maximum olding o he o ic epi helium occu s (Ál a ez and Na ascues 1990) and he e is also close con ac be ween he basal laminae o he medial pa o he o ic pi and he neu al ube. This appea s o be ela ed o o ic placode in agina ion, since basal lamina HSPG deg ada ion by mic oinjec ion wi h hepa inase III al e s o ic epi helium olding. The absence o Ac. an i-hepa an sulpha e immunoma king on he basal lamina o he ea ed o ic p imo dium shows he e ec i eness o ea men wi h hepa inase. The in eg i y o he ex acellula ma ix and he basal lamina seems necessa y o no mal mo phogenesis o he inne ea , and ce ain s udies suppo he hesis ha glyco- p o eins may play an impo an ole in o ic placode in ag- ina ion. T ea men o he o ic p imo dium wi h unicamy- cin (Rausch and Hil e 1988), a d ug which inhibi s N- linked glycosyla ion (Mahoney and Duskin 1979), dis- up s placode in agina ion. Mic oinjec ion o β-D-xylo- side in he ex acellula ma ix benea h he o ic placode causes delayed in agina ion in chick (Ge chman e al. 1995) and a (Alonso e al. 1998, 1999) emb yos, his d ug dis up s he syn hesis o sulpha ed p o eoglycans, pa icula ly chond oi in sulpha e p o eoglycan and HSPG (Sobue e al. 1987; Lugemwa and Esko 1991; Lugemwa e al. 1996). I has also been demons a ed ha enzyma ic deg ada ion o ex acellula ma ix hyalu onic acid dis- up s o ic epi helium olding (Ge chman e al. 1995). P e- iously men ioned da a and he p esen esul s sugges ha glycop o ein molecules and especially p o eoglycans play an impo an ole in o ic placode in agina ion, bu he mechanism by which hese molecules may con ol epi he- lial olding is no clea . Hyalu onic acid and sulpha e p o eoglycans a e known o possess g ea hyd ophylic ca- paci y (Compe and Zampa o 1990) and can b ing abou an inc ease in in e cellula spaces in he ex acellula ma- ix ha could exe a mechanical o ce on he edges o he o ic epi helium and acili a e i s olding. In his ega d we know ha hyd a ion caused by hyalu onic acid and p o eoglycans is an impo an ac o in neu oepi helium olding in he o ma ion o he neu al ube (Schoenwol and Fishe 1983; Mo iss-Kay and Tucke 1989). A desc ip ion has also been gi en o he p esence o adhesion molecules in he o ic epi helium du ing in agi- na ion, mo e speci ically N-CAM and L-CAM (Richa d- son e al. 1987); he o me appea s h oughou he whole o he epi helium bu is mo e concen a ed in he la e al po ion, whils L-CAM has a uni o m dis ibu ion pa e n. Recen ly B own e al. (1998) demons a ed ha in a ian emb yos mic oinjec ion o blocking an ibodies o N- CAM in o he pa axial mesode m adjacen o he o ic pla- code esul ed in wo majo classes o de ec s: de achmen o he p imo dium om he neu al ube and in e e ence wi h o ma ion o he o ic olds. I is known ha HSPG may in e ac wi h N-CAM a plasma ic memb ane le el (Cole and Bu g 1989) and his in e ac ion migh p oduce changes in N-CAM s uc u e ha modula e cellula adhe- sion, he eby acili a ing epi helial olding. I has also been sugges ed ha cell su ace p o eogly- cans migh es ablish connec ions be ween he in acellu- la cy oskele on and he ex acellula ma ix (Goe inck 1991; Jalkanen e al. 1991), which could b ing abou changes in he shape o he cells ha would condi ion he olding o he epi helium. Howe e , as was poin ed ou in he in oduc ion, he ole o he cellula cy oskele on ap- pea s o be o li le ele ance in o ic placode in agina ion. HSPG is known o be capable o joining wi h di e - en componen s o he ex acellula ma ix ia i s chains o glycosaminoglycan (Goe inck 1991). These in e ac- ions be ween HSPG and he ex acellula ma ix migh 340 explain he ac ha enzyma ic diges ion o he HSPG in- hibi s o ic placode in agina ion, and i would cons i u e a poin in common wi h al e a ions in o ic p imo dium olding induced by he al e a ion o o he componen s o he ex acellula ma ix. The HSPG may join wi h he i- b onec in (Toole 1991; Paulsson 1992), which in u n is capable o joining wi h he collagen IV (Yamada 1985); hese molecules o m pa o he o ic placode basal lami- na du ing i s in agina ion and, in ac , con ibu e o hind- b ain neu oepi helium and o ic epi helium ancho ing (Hil e and Randolph 1993). This ancho ing appa en ly plays an impo an ole in epi helial olding, since i could s abilise a pa o he epi helium whils in o he e- gions ac i e o passi e mo emen s occu ha lead o epi- helial in agina ion. Enzyma ic diges ion o he HSPG could de-s abilise hese connec ions be ween he o ic epi helium and he neu al ube and his migh accoun o he dis up ion in in agina ion induced by ea men wi h hepa inase. Wha is mo e, we ha e obse ed ha al- hough dis up ion o in agina ion is gene alised, i is mo e isible in he medial pa o he o ic placode, and ha he e is sepa a ion be ween o ic and neu al epi helia. These ac o s suppo he hypo hesis conce ning he im- po ance o o ic placode basal lamina HSPG in i s union wi h he hindb ain neu oepi helium. I has also been pos ula ed ha a di e en ial g ow h o he o ic p imo dium could b ing abou in e nal o ces wi hin he epi helium leading o i s in agina ion (Ál a ez and Na ascues 1990), as occu s in he neu oepi helium du ing ele a ion o he neu al olds (Sausedo e al. 1997) o in lung-b anching mo phogenesis (Gallaghe 1986). In his ega d HSPG migh s imula e epi helial g ow h on accoun o i s associa ion wi h g ow h ac o s. I is known ha HSPG can bind o se e al g ow h ac o s, modula ing i s ac i i y (Goe inck 1991; Ruoslah i and Yamaguchi 1991; A ieze e al. 1994a,b; Johnson and Wong 1994; Schuge e al. 1996; Da id and Be n ield 1998). This in- e ac ion has been s udied mainly o ib oblas g ow h ac o s (FGFs) and se e al mechanisms ha e been p o- posed (Jaakkola and Jalkanen 1999) o explain how HSPG may modula e he ac ion o he FGFs. Fi s , he glycosaminoglycans in p o eoglycans may induce oligo- me iza ion o FGF molecules equi ed o ib oblas g ow h ac o ecep o clus e ing. Second, p o eoglycans could p o ec FGFs om p o eoly ic deg ada ion and se e as a ese oi o hese hepa in-binding g ow h ac- o s. Thi d, HSPG could simply se e as molecules ha ha e an abili y o p esen he FGFs o he high-a ini y e- cep o s. Finally, i has been hypo hesised ha he main unc ion o p o eoglycans is o educe he dimensionali y o FGF di usion. In addi ion, i has been p o en ha HSPG enzyme deg ada ion o ea men wi h hepa an sul- pha e an ibody inhibi s lung-b anching mo phogenesis in mouse emb yos and ha addi ion o basic ib oblas g ow h ac o – a ypical hepa in-binding g ow h ac o – es o ed he inhibi ion caused by hepa i inase (Schuge e al. 1996; To iyama e al. 1997). As ega ds o ic p imo dium de elopmen , i has been shown ha di e en sec e ed ac o s like he BMP- amily o TGFβ-like polypep ides, FGF-2 and FGF-3 a e ex- p essed in he o ic placode and o ic esicle (Oh e al. 1996; Wu and Oh 1996; Mahamood e al. 1995, 1996; To es and Gi áldez 1998). I is no clea whe he hese ac o s can ha e an e ec in o ic placode in agina ion, al- hough i has been sugges ed ha FGFs migh play a ole in his p ocess. This hypo hesis was suppo ed by he ob- se a ion ha o ocys o ma ion in chick emb yos was blocked by an i-sense oligonucleo ides o FGF-3 p o ein and by an ibodies agains FGF-3 in explan cul u e (Re- p esa e al. 1991). Howe e , mice homozygous o a a - ge ed dis up ion o he FGF-3 gene o m o ocy s ha a e no mal in mo phology bu whose subsequen mo phogen- esis and di e en ia ion a e abe an (Mansou e al. 1993). Simila ly, in he K eisle mu an mouse, in which FGF-3 is exp essed a e y low le els (F ohman e al. 1993; McKay e al. 1994), he o ocys o ms bu ails o unde go no mal mo phogenesis and di e en ia ion (Deol 1964). These s udies appea o indica e ha FGF-3 is es- sen ial o la e s ages o o ic mo phogenesis and di e en- ia ion bu no o in agina ion o he o ic placode. Since, as we ha e al eady men ioned, HSPG may modula e he ac i i y o g ow h ac o s ha could ha e an in luence on o ic p imo dium de elopmen , we ha e made an analysis o apop osis, cell p oli e a ion and he size o he o ic epi helium. Cellula apop osis de ec ed by he TUNEL me hod was obse ed solely on he la e al edge o he o ic pi on he side ea ed wi h hepa inase III, and in he usion and closu e a ea o he o ic opening on he con ol side. Bo h hese a eas o apop osis may be conside ed physiological as hey appea du ing no mal de elopmen o he o ic p imo dium (Ma o i z e al. 1977; Rep esa e al. 1990). Analysis o o ic epi helium size showed no s a is ically signi ican di e ences be- ween he ea ed and con ol sides; simila ly, al hough he e was a sligh educ ion in he numbe o B dU s ained cells on he ea ed side, his di e ence was no s a is ically signi ican . This sugges s ha enzyme- deg ading o basal lamina HSPG does no in e e e wi h he su i al o p oli e a ion o cells o wi h o ic epi helium g ow h; howe e , we canno ule ou he co-pa icipa ion o HSPG and g ow h ac o s in o ic placode in agina- ion, which migh ac in a di e en way om ha o egula ion o cell p oli e a ion. To conclude, his s udy p o ides expe imen al e i- dence ha he in eg i y o basal lamina componen s is a equisi e o no mal o ic placode in agina ion, despi e he ac ha we canno exclude he co-in e en ion o o he ac o s in o ic epi helium olding. Re e ences Alonso MI, Ga o A, Mo o JA, Ma ín P, Ba bosa E (1998) Neu al ube de ec s induced by β-D-xyloside: e idence o a ole o sul a ed p o eoglycans in neu al old usion in a emb yos. Eu J Ana 2:133–140 Alonso MI, Ga o A, Mo o JA, Ma ín P, Ba bosa E (1999) In- ol emen o sul a ed p o eoglycans in emb yonic b ain ex- pansion a ea lies s ages o de elopmen in a emb yos. Cells Tissues O gans 165:1–9 341