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