PERdev91a.pdf

Development

HI,

583-599 (1991)

Printed

in

Great Britain

©

The

Company

of

Biologists Limited

1991

583

Spatial and temporal changes

in the

distribution

of

proteoglycans during

avian neural crest development

ROBERTO PERRIS

1

*, DANUTA KROTOSKI

2

, THOMAS LALLIER

1

, CARMEN DOMINGO

3

, J.

MICHAEL SORRELL

4

and MARIANNE BRONNER-FRASER

1

'Developmental Biology Center, University

of

California, Irvine,

CA

92717,

USA

2

National Institute

of

Child Health

and

Human Development, Executive Plata North, Room 643,

6130

Executive

Blvd.,

Rockville,

MD

2085,

USA

^Department

of

Cell

and

Molecular Biology, University

of

California, Berkeley,

CA

94720,

USA

4

Department

of

Biology, Case Western Reserve University, Cleveland,

OH

44106,

USA

*

Author

for

correspondence

Summary

In this study,

we

describe

the

distribution

of

various

classes

of

proteoglycans

and

their potential matrix

ligand, hyaluronan, during neural crest development

in

the trunk region

of

the chicken embryo. Different types

of chondroitin

and

keratan sulfate proteoglycans were

recognized using

a

panel

of

monoclonal antibodies

produced against specific epitopes

on

their glycosamino-

glycan chains.

A

heparan sulfate proteoglycan

was

identified

by an

antibody against

its

core protein.

The

distribution

of

hyaluronan was mapped using

a

biotinyl-

ated fragment that corresponds

to the

hyaluronan-

binding region

of

cartilage proteoglycans. Four major

patterns

of

proteoglycan immunoreactivity were

ob-

served. (1) Chondroitin-6-sulfate-rich proteoglycans and

certain keratan sulfate proteoglycans were absent from

regions containing migrating neural crest cells, but were

present in interstitial matrices and basement membranes

along prospective migratory pathways such

as the

ventral portion

of the

sclerotome. Although initially

distributed uniformly along

the

rostrocaudal extent

of

the sclerotome, these proteoglycans became rearranged

to

the

caudal portion

of

the sclerotome with progressive

migration

of

neural crest cells through

the

rostral

sclerotome

and

their aggregation into peripheral

ganglia.

(2) A

subset

of

chondroitin/keratan sulfate

proteoglycans bearing primarily unsulfated chondroitin

chains was observed exclusively

in

regions where neural

crest cells were absent

or

delayed from entering, such

as

the perinotochordal

and

subepidermal spaces.

(3) A

subset

of

chondroitin/keratan sulfate proteoglycans

was

restricted

to the

perinotochordal region

and,

following

gangliogenesis,

was

arranged

in a

metameric pattern

corresponding

to the

sites where presumptive vertebral

arches form.

(4)

Certain keratan sulfate proteoglycans

and

a

heparan sulfate proteoglycan were observed

in

basement membranes

and in an

interstitial matrix

uniformly distributed along

the

rostrocaudal extent

of

the sclerotome. After gangliogenesis,

the

neural crest-

derived dorsal root

and

sympathetic ganglia contained

both these proteoglycan types,

but

were essentially free

of other chondroitin/keratan-proteoglycan subsets.

Hyaluronan generally colocalized with

the

first

set of

proteoglycans,

but

also

was

concentrated around

mi-

grating neural crest cells

and was

reduced

in

neural

crest-derived ganglia. These observations demonstrate

that proteoglycans have diverse

and

dynamic distri-

butions during times

of

neural crest development

and

chondrogenesis

of the

presumptive vertebrae.

In gen-

eral, chondroitin/keratan sulfate proteoglycans

are

abundant

in

regions where neural crest cells are absent,

and their segmental distribution inversely correlates

with that

of

neural crest-derived ganglia.

Key words: proteoglycans, hyaluronan, neural crest, avian

embryo, cell migration, extracellular matrix.

Introduction

Neural crest cells migrate long distances along pathways

containing an intricate extracellular matrix (ECM). As

a consequence, the ECM is thought to play a central

role in several aspects of neural crest development.

In

vitro,

neural crest cells migrate avidly on numerous

ECM molecules including fibronectin, laminin and

collagens (Newgreen and Erickson, 1986; Perris and

Johansson, 1987; Perris

etal.

1989; Perris

et

al.

1990a),

suggesting that individual matrix components may serve

as permissive migratory substrates. Ultrastructural

studies performed

in situ

reveal that neural crest cells

form specialized contacts with this fibrillar matrix

584

R. Ferris and others

network encountered during migration (Lofberg

et al.

1980;

Newgreen and Erickson, 1986; Penis

et al.

1990b).

In ovo

injections of antibodies against indi-

vidual matrix molecules or their cell surface receptors

results in abnormal neural crest development

in vivo

(Bronner-Fraser, 1985, 1986a; Bronner-Fraser and

Lallier, 1988; Bronner-Fraser, 1988). Moreover, trans-

plantations of regionally and temporally denned

matrices adsorbed onto membrane microcarriers have

provided evidence that the ECM can prematurely

promote the onset of neural crest cell movement

in vivo

(Lofberg

et al.

1985, 1988).

A logical first step in establishing the role of the ECM

in neural crest cell migration is to determine its

structural and molecular composition at various phases

of neural crest development. Ultrastructural and

immunohistochemical studies have revealed that the

interstitial matrix along trunk neural crest migratory

pathways consists of a fibrillar collagenous network,

which contains abundant amounts of fibronectin,

tenascin/cytotactin and glycosaminoglycans (Newgreen

and Erickson, 1986; Perris and Bronner-Fraser, 1989;

Perris

et al.

1990a; Newgreen

et al.

1986, 1990).

Basement membrane matrices, enriched in laminin and

collagen type IV, also line some neural crest migratory

routes (Newgreen and Erickson, 1986; Perris and

Bronner-Fraser, 1989; Perris

et al.

19906).

Although a great deal of information has been

compiled regarding the distribution and possible func-

tion of cell adhesion glycoproteins such as fibronectin,

laminin, cytotactin/tenascin and various collagens

during neural crest cell migration, far less is known

about the role of proteoglycans in this process.

Proteoglycans represent a heterogeneous population of

molecules that contribute to the compositional diversity

of the ECM. Indirect evidence that proteoglycans might

be well-represented during neural crest development

has emerged from a series of histochemical studies

using cationic dyes,

in situ

metabolic labelling and

differential enzymatic degradation (Kvist and Finne-

gan, 1970; Pintar, 1978; Lofberg

et

al.

1980; Newgreen

et al.

1982, 1986; Perris

et al. \99Qb).

From these

observations it was concluded that several distinct

families of matrix and cell-associated proteoglycans

were expressed at various phases of neural crest

development. However, the nature and spatiotemporal

distribution of specific populations of proteoglycans

present at these early stages of development, as well as

their relationship to their potential ligands, such as

collagens and hyaluronan, has not been determined.

In this study, we have examined the distribution of

various proteoglycan subclasses

in situ

and have

determined their spatial and temporal organization

relative to the development of the trunk neural crest in

the chick embryo. For this purpose, a panel of

monoclonal antibodies that specifically detect native

carbohydrate structures of chick proteoglycans were

used in combination with differential enzymatic degra-

dation and antibodies generated against chondroiti-

nase-digested murine proteoglycans. In addition, a

specific probe for hyaluronan (Knudsen and Toole,

1985;

Ripellino

et al.

1985) was used to map the

distribution of this potential matrix and cell surface-

associated ligand for proteoglycans. Our observations

indicate that a time-related and region-specific organiz-

ation of diverse types of proteoglycans occurs during

neural crest cell migration and gangliogenesis, and the

onset of vertebral chondrogenesis.

Materials and methods

Production and

screening

of monoclonal antibodies

(mAbs)

against

chondroitin (CS) and keratan sulfate

(KS)

chains

of

proteoglycans

(PGs)

Native CS/KS-PG monomers were extracted from bone

marrow of femurs and tibias of

17

day old chick embryos using

4 M

guanidine-HCl in the presence of protease inhibitors as

previously described (Oegema

et

al.

1975; Sorrell

et

al.

1988).

Solid CsCl was added to the extract to give the final density of

1.5gml"

1

,

and the solution was centrifuged at 100000 g for

48

h. The bottom one third of the gradient, with the density of

1.6gml"',

was collected, dialyzed against

0.15

M

NaCl, then

water and lyophilized as previously described (Sorrell

et al.

1988).

The purified, high buoyant density PGs (Dl fraction),

which have been shown to be substituted with both

CS

and KS

chains, were then used to immunize mice. The immunization

protocol and the protocol for the production of hybridoma

were as previously published (Caterson

et

al.

1987; Sorrell

et

al.

1990). Clones resulting from the fusion were tested in

ELISA for the presence of antibodies that recognized the

marrow PGs as well as PGs from other sources. Antibodies

produced by nine such clones, 7D4, 6C3, 4C3, 4D3, 3D2,

1B4,

4D1,

2D3 and 8C2, were identified and classified as IgM

kappa. Each clone was then subcloned and used to produce

ascites fluid. The ascites fluid was further screened for the

ability of the antibody to identify epitopes on a number of

different purified CS/KS-PG monomers, in their intact form

or after alternative digestion with chondroitinase ABC and

keratanase (see below).

Other

antibodies

MAbs 2B6, 1B5, 3B3 and 5D4 were obtained from Dr Bruce

Caterson, Division of Orthopaedics, Department of Surgery,

University of North Carolina, Chapel Hill, NC. The mAb

33-2,

kindly provided by Dr Douglas Fambrough (The John

Hopkins University, Baltimore, MA), reacts with a core

protein-associated epitope located on a large molecular mass

HS-PG (MW>300xlO

j

M

r

) of embryonic chick skeletal

muscle (Bayne

et

al.

1984). The HNK-1 antibody was purified

from ascites fluid (American Tissue Culture, Inc) by column

chromatography on protamine sulfate followed by ammonium

sulfate precipitation. In some cases, the purified HNK-1

antibody

was

biotinylated using biotin-A'-hydroxysuccinamide

ester (Calbiochem) according to a previously published

procedure (Perris and Johansson, 1987; Perris

et

al.

1989).

ELISA

Proteoglycan monomers from shark cranial cartilage (Al),

embryonic chick epiphysial cartilage (Dl), bovine articular

cartilage (A1D1) bovine nasal cartilage (A1D1) and Swarm

rat chondrosarcoma (A1D1) were purified according to

previously published procedures (Oegema

et

al.

1975; Sorrell

et

al.

1988, 1990). Wells of microtiter plates (Costar half area

plates) were coated with PG monomers 25-50 ^g

ml"

1

(dry

weight) in PBS overnight at 4°C. Proteoglycan-free areas of

the wells were then blocked with

1

% BSA (v/w) in PBS for

Proteoglycans in avian neural crest development

585

1-2

h

at 37°C. In some experiments, wells with immobilized

PGs were digested for 5min at room temperature with

lO^i.u. mP'-lOOmi.u. ml"

1

chondroitinase ABC (Sigma), or

70mi.u.ml~'

Pseudomonas

endo-/5-galactosidase (keratan-

ase;

ICN Biochemicals Inc.), dissolved in Tris-HCl buffer,

pH7.6,

containing protease inhibitors and 1% BSA.

Digested and untreated proteoglycan-coated wells were

extensively rinsed with cold PBS and incubated with the

various mAbs (1:100-1:700000) diluted in PBS for lh at

37

°C,

followed by sequential incubation with horseradish

peroxidase-conjugated goat anti-mouse Ig (Southern Biotech-

nology) diluted 1:500 in 0.1

M

Tris-0.9

%

NaCl pH7.6 for

1

h

at 37°C, and o-phenylenediamine (0.4mgml~')/H2O2

(0.0015%). The enzyme reaction was stopped by addition of

2 M

H

2

SO

4

and the adsorbance readings were performed at

492 ran in a Bio-Rad microplate reader. Competitive ELISA

was carried out by coating microwell plates with 50/igml"

1

monomers of shark cranial cartilage proteoglycan as indicated

above. Serial 50% dilutions of shark cranial cartilage, chick

bone marrow, chick epiphysial cartilage, bovine articular

cartilage or bovine nasal cartilage were prepared in PBS with

1

%

BSA. Equal aliquots of diluted antigen, or buffer alone,

were added to a constant volume of antibody (6C3 diluted

1:25000;

7D4 diluted 1:150000; 4C3 diluted

1:6000)

in the

same buffer, incubated at 37°C for

1

h, and then added to the

coated wells. Standard ELISA was performed as above. The

% inhibition was determined for three assays, averaged and

plotted

versus

/igml"' of competing antigen. The amount of

competing antigen required to give

50 %

inhibition was then

obtained by extrapolation from the inhibition curves.

Preparation of the hyaluronan probe

A 65xlO

3

/W

r

proteolytic fragment corresponding to the

hyaluronan-binding region of the bovine nasal cartilage

proteoglycan was generously provided by Dr Torward

Laurent (Department of Medical and Physiological Chemis-

try, Biomedical Center, Uppsala, Sweden). The fragment was

generated by affinity chromatography as previously described

(Tengblad, 1979; Laurent and Tengblad, 1980). The PG

fragment, with attached low molecular weight hyaluronan

(16—30 oligosaccharides) to preserve the hyaluronan-binding

activity, was biotinylated according to the same procedure as

indicated for the HNK-1 antibody. Purified hyaluronan

(human umbilical cord;

M

T

1000000; Sigma) was bound to

AH-Sepharose 4B (Pharmacia) as described by Tengblad

(1979).

Before use, the biotinylated hyaluronan-binding

fragment was then dissociated from the hyaluronan oligosac-

charides by transfer to a

4M

guanidine-HCl-50mM sodium

acetate buffer, pH5.8, containing

10 mM

EDTA,

100 mM

6-aminohexanoic acid, 5mM benzamidine hydrochloride, and

2mM phenylmethylsulfonyl fluoride (PSMF; Sigma).

Preparation of tissues for immunohistochemistry

Chick embryos at developmental stages 14-23 were rapidly

frozen in liquid nitrogen-cooled isopentane and immediately

transferred to methanol precooled to — 80°C and stored at this

temperature for 3 days. They were then successively

transferred to -35°C for

1

day, -20°C for 2 days and 4°C for

2 days. Following cryofixation, embryos were embedded in

paraplast after one change of methanol:histosol (7:3), one

change of methanol:histosol (3:7), two changes of histosol,

one change of histosol:paraplast (1:1), and three changes of

paraplast at 20min for each step. A number of other

procedures, including direct fixation in modified Karnovsky's

fixative,

4%

paraformaldehyde, Sainte-Marie, periodate-

lysine-paraformaldehyde and Zenker's fixatives followed by

paraplast embedding or cryosectioning were also tested and

were found to be significantly less efficient in retaining

reactivity for the various anti-proteoglycan mAbs. One

exception was the mAb 33-2 (anti-HS-PG antibody) for

which optimal preservation of immunoreactivity was obtained

after methanol fixation and cryosectioning. The latter was

accomplished according to previously published procedures

(Krotoski

et al.

1986). Serial transverse, horizontal and

parasagittal sections through the mid-trunk level of the

embryo were cut at 10-20

um

and mounted on gelatinized or

albuminized slides.

Enzymatic pre-digestions of tissue sections

In cases where antibody application was preceded by

enzymatic digestion, sections were incubated with the various

enzymes for 1-3h at 37°C.

Streptomyces

hyaluronidase

(Sigma) was used at 115i.u.ml~' in

0.1M

sodium acetate

buffer, pH5.2, containing 250^gml~

1

ovomucoid (trypsin

inhibitor O-IV),

1 mM

PMSF,

1 mM

EDTA,

1 mM

iodoacetida-

mine and 200Ki.u. ml"

1

aprotonin (all Sigma). Chondroiti-

nases ABC and AC II (Sigma) were used at 0.2i.u. ml"

1

in

0.1M

bicine buffer (Calbiochem), pH8.0, containing 0.1%

BSA. Keratanase

{Pseudomonas

sp. IFO 13309) and endo-/S-

galactosidase

(Escherichia

freundii;

ICN Biochemicals Inc.)

were applied at O.Oli.u.ml"

1

in

50 mM

Tris-HCl buffer,

pH7.2,

containing 80mM NaCl,

1 mM

EDTA,

1 mM

iodoaceta-

mide,

1 mM

PSMF, and 5^gml~' pepstatin A (Sigma).

Immunohistochemistry

Incubation with primary antibodies (1:50-1:150 in

0.01M

phosphate buffer, pH7.2, containing 0.1

%

BSA) was carried

out overnight at 4

C

C, except for the HNK-1 labelling that was

performed for 2h at room temperature. Antibody-antigen

binding was visualized by indirect immunofluorescence using

Ig-class and species specific secondary antibodies, directly

conjugated to fluorescein, or rhodamine (Zymed Labora-

tories).

Double labelling was performed using biotinylated

HNK-1 antibody, visualized by incubation of the sections with

streptavidin-Texas Red complexes (1:200; Amersham).

Labelled sections were mounted in glycerol-Tris-HCl buffer,

pH8.0,

containing 2mgml"

1

l,4-diazabicyclo(2,2,2,)octane.

In some cases, comparative stainings with anti-PG antibodies

and the HNK-1 antibody were carried out on adjacent

sections to avoid possible steric hindrance effects of IgM

antibodies.

Detection of hyaluronan in tissue sections

Stage 14-23 embryos were fixed in phosphate-buffered 4%

paraformaldehyde, pH7.2, containing 0.5% cetylpyrinidium

chloride and 0.1% polyvinylpyrrolidone (M

r

40000; Sigma)

for 12-18

h

at

4

(Derby and Pintar, 1978). Fixed embryos were

rinsed, embedded in gelatin and sectioned as described

above. Tissue sections were incubated with 30-100

/.ig

biotinylated hyaluronan probe in

0.1M

phosphate-buffered

saline, pH7.2, at 4°C overnight. Sections were then

extensively rinsed in buffer containing 0.01 % Nonidet P-40,

incubated with streptavidin-fluorescein and finally mounted

as described for antibody-labelled sections. Control sections

were predigested with

Streptomyces

hyaluronidase (Sigma) in

the presence of protease inhibitors (see above), or incubated

with inactivated probe. For the latter purpose, the biotinyl-

ated hyaluronan-binding fragment was preincubated with a

molar excess (100

ng

ml"

1

) of hyaluronan oligosaccharides

(16-30) for

1

h at 37°C, dialyzed to remove excess nonbound

oligosaccharides, and then applied to the sections. In some

cases,

sections were double-labelled with the hyaluronan

probe and the HNK-1 antibody.

586

R.

Ferris

and

others

Confocal laser microscopy

In

a

number

of

cases, stained sections were analyzed

by

confocal laser microscopy

in

conjunction with computerized

image analysis. For this purpose, we used a Bio-Rad MRC 500

laser unit attached

to an

epifluorescent microscope (Nikon,

Labphot),

a

videoprinter VP 3500 (Seikosha),

a

3D6 personal

computer,

and

Bio-Rad software

for

image analysis. Relative

staining intensities were analyzed according

to an

arbitrary

value scale

of

0-255

in a

series

of 74

transversal sections

cut

between somite

15

and

22

of

stage

17

embryos (29-31 somites)

and stained with

mAb 4C3; and a

series

of 16

horizontal

sections spanning

>20

somites

of

similar

age

embryos stained

with

mAb 7D4.

Transverse sections were analyzed

in the

medial-lateral plane

by

single spot measurements across their

dorsal portion, starting

and

ending

in the

subepidermal space.

A single spot measurement

was

also taken within cells

of the

neural tube where no immunoreactivity was observed

and

was

used

to

correct

for the

background nonspecific fluorescence.

Analysis

in the

dorsal-ventral dimension

was

carried

out by

consecutive single spot measurements along

an

arbitrary

vertical line drawn across

the

section

and

passing through

the

subepidermal space,

the

lateral sclerotome,

the

perichordal

area

and

finally

the

periaortic area. Horizontal sections

containing

the

mid-portion

of the

neural tube were analyzed

in

the

rostral-caudal dimension

by

recurring spot measure-

ments

at the

borders

and

center

of

each somite, and within

the

intersomitic clefts, starting from

the

most recently formed

somite

and

progressing rostrally.

In

this case, control values

for correction

of the

nonspecific background fluorescence

were obtained from

the

dermamyotome, which consistently

lacked proteoglycan immunoreactivity.

Specificity

of

the

anti-chondroitin/keratan sulfate

mAbs

The characteristics

and

specificities

of the

various mAbs

are

summarized

in

Table

1.

Immunization

of

mice with high

buoyant density

PGs

extracted from bone marrow

of

17 days

old chick embryos resulted

in

nine clones producing mAbs

specific

for CS and

KS glycosaminoglycans. MAbs 7D4,

6C3,

4D3

and

4C3

all

reacted with epitopes expressed

on

native

CS

moieties

of

cartilage

PGs

from

a

variety

of

sources including

chick epiphysis, bovine arteries

and

nose, chick bone marrow

and shark cranium

(Fig. 1).

Although

all

five mAbs detected

epitopes

on

CS-PGs, direct

and

competitive ELISA using five

distinct

PG

types

as

model antigens clearly demonstrated that

each antibody detected

a

different structural element

(Fig.

1A).

ELISA also showed that, while

the

antigenic

profiles

of the

chick CS-PGs were related,

the

antigenic

profiles

of

the other CS-PGs were characteristic

for

each type.

Combined

use of

ELISA

and

chondroitinase

ABC

digestion

proved that

the

immunological diversity resided

in the CS

chains. Specific epitopes were differentially removed

by

various concentrations

of

enzyme

(Fig. IB),

consistent with

the idea that each

of

these antibodies recognized

a

unique

structural unit.

For

example,

the

epitope recognized

by mAb

6C3

was

ten-fold more sensitive

to

digestion than those

recognized

by 7D4 or

4C3. Recent data indicate that mAbs

7D4,

6C3 and 4C3

primarily recognize

CS

chains with

prevalent 6-sulfated groups, whereas mAb 4D3 seems

to

react

primarily with 4-sulfated chondroitins (Sorrell, unpublished).

MAbs

4D1, 3D2, 1B4, 8C2 and 5D4

also recognized

epitopes

on

CS-PG monomers, biit unlike

the

five mAbs

described above, these epitopes were

not

removed

by

chondroitinase predigestion

of the

antigens.

In

fact, anti-

genicity

of PGs for the

latter mAbs sometimes increases

following chondroitinase treatment (data

not

shown).

In

Table

1.

Specificity

and

characteristics

of

the

various

mAbs

Antibody

7D4

6C3

4C3

4D3

3B3

1B5

2B6

4D1

3D2

8C2

5D4

33-2

Antigen

CS-PG

(CS)/KS-PG

HS-PG

Reactivity

native CS

Ch6S

ChO

Ch4S/DS

native KS

>300kD CP

Epitope

unknown

GlcUA-6SO

4

, /V-AcGal

GlcUA, /V-AcGal/Glu

GlcUA-4SO

4

, JV-AcGal

unknown

(N-AcLac)

n

»fi,SO4

unknown

Note:

The epitopes recognized by mAbs 1B5, 3B3 and 2B6 are

generated by chondroitinase ABC/AC II digestion (Couchman

et

al.

1984; Caterson

et al.

1987), which involves an elimination

reaction introducing a

4,5-delta

unsaturation of the GlcUA moiety

of glycosaminoglycan disaccharides. The epitope recognized by

mAb 5D4 comprises a miminum of six sugar units in linear

nonsubstituted sulfated poly-(A'-acetyllactosamine) structures of KS

(Mehmet

et al.

1986; Caterson

et al.

1987). mAb 2B6 has a dual

reactivity for both Ch4S and DS chains substituted on

proteoglycan core proteins. Its specificity is determined by

chondroitinase ABC or AC II digestion prior to immunostaining.

Following chondroitinase ABC digestion the mAb recognizes both

Ch4S and DS, whereas following chondroitinase AC II digestion it

reacts only with Ch4S. The mAb 3B3 also detects an unidentified

epitope on mature chick cartilage proteoglycans that is not

generated by chondroitinase digestion (Sorrell

et al

1990). GlcUA,

glucuronic

acid;

N-AcGal/Glu, JV-Acetylgalactosamine/

glucosamine; iV-AcLac, yV-Acetyllactosamine; CP, core protein.

contrast, digestion

of

immobilized embryonic chick cartilage

CS-PG abolished virtually

all

immunoreactivity

for

these

mAbs

(Fig. 1C). The

moderate reduction

in

immunoreac-

tivity

for

bovine nasal cartilage CS-PG

is

likely

to be due to

differences

in the

structure

of the KS

chains

of

these

two

species-specific

PGs.

The overall staining patterns obtained with mAbs 7D4,

6C3

and 4C3 were similar

and

therefore

are

described collectively.

However,

the

extent

and

intensity

of the

labelling obtained

with these mAbs decreased

in the

order 4C3>7D4>6C3,

even though

mAb 7D4

reacted

15

times more strongly with

isolated chick proteoglycans than mAb 4C3 (Fig.

1A).

Minor

differences

in the

immunoreactive patterns obtained with

these mAbs were also observed within

the

sclerotome

and in

the perinotochordal area.

Results

Analysis

of

the distribution

of

proteoglycans (PGs)

and

hyaluronan

was

carried

out in the

mid-trunk region

at

two developmental stages:

during neural crest cell

migration

(stages

14-19 at

level

of

somites 12-27;

Fig.

2A) and

following gangliogenesis

(stages

21-23

between wing

and

limb buds). Four generalized

patterns

of

immunoreactivity were distinguishable

and

those will

be

referred

to as

patterns

I-IV

(Table

2).

Distribution

of

chondroitin sulfate proteoglycans

(CS-

PGs)

During neural crest cell migration

(stages

14-19)

Proteoglycans detected

by the

mAbs 7D4/6C3/4C3

o

/>

CD

3

CD-6C3 ED-4C3

=7D4

10.00-

1.00-

0.10-

0.01

•

1.O0E-3-

1.00E-4

J

CBM

CEC

SCC

BAC

BNC

Type

of

CS-PG

100.00

T

eo.oo-

40.00-

•

20.00

••

0.00

10

,-2

10

,-1

B

Concentration chondrortinase

ABC

(U/ml)

K3

•

CEE CS—PC

M -CEECS-PC

+

luratoix—

CD

-

BNC CS-PG

KB -BNCCS-P0

+

karatanaM

1.500

1.200-

0.8OO-

0.300-

0.000

5D+

3D2

401

BC2

Antibody

were distributed widely along neural crest cell

mi-

gratory pathways.

The

immunostaining patterns

pro-

duced

by

antibodies 7D4/6C3/4C3

(Fig. 2B)

were

similar

to

that

of

mAb 3B3, which recognizes chondroi-

tin-6-sulfate (Ch6S) moieties following chondroitinase

ABC digestion. Thus,

it is

possible that

the

native

epitopes recognized

by

these antibodies were expressed

on

CS

chains rich

in

6-sulfated groups. This staining

pattern will

be

refered

to as

pattern

I

(Table 2).

At

initial stages

of

neural crest cell migration, pattern

I

CS-

PGs were detected within

the

subepidermal basement

Proteoglycans

in

avian neural crest development

587

Fig.

1. (A)

Competitive ELISA using shark cranial

'

cartilage

(SCC)

proteoglycan

as an

immobilized antigen

and chick bone marrow (CBM),

CEE,

bovine articular

cartilage

(BAC) and BNC

protoglycan monomers

as

competing antigens. Values denote means

of the

amount

of

competing proteoglycan yielding

50 %

inhibition

of the

antibody-antigen binding.

The

values

are

expressed

as

l/amount

of

competing proteoglycan

and

hence

a

high

value indicates that

the

proteoglycan

was an

efficient

competitor

of the

antibody-antigen binding.

MAb 6C3

recognizes

an

epitope

on BNC

CS-PG,

but 50%

inhibition

level

was not

reached using

up to

1000

/.ig

ml"

1

competing

antigen

(*).

Because

of its low

affinity

for SCC

proteoglycan,

mAb 4D3

could

not be

tested

in

this assay.

(B) Concentration-dependent effects

of

chondroitinase

ABC digestion

of

immobilized

CEE

proteoglycan

monomers

on the

binding

of the

various mAbs.

MAb 2B6

was used

as a

control

for the

efficiency

of the

enzymatic

degradation, since digestion with chondroitinase

ABC is

required

to

generate

the

epitope recognized

by

this

antibody

(see

Table

I). The

data indicate that

CS

chains

are degraded

at

enzyme concentrations higher than

10~

4

i.u. ml"

1

. Note that

the

epitopes recognized

by

mAbs

6C3,

4C3 and 7D4 are

totally removed

by the

chondroitinase digestion,

but

only

by

enzyme

concentrations considerably higher than those needed

to

generate

the 2B6

epitope. Each point corresponds

to %

values

of the

maximal adsorbance readings from triplicate

tests.

(D)

Differential elimination

of

antibody reactivity

following keratanase digestion

of CEE and BNC

proteoglycan monomers (mean values

of

maximal

adsorbance).

The

differential reduction

in

antigenicity seen

for

CEE

versus

BNC

CS-PG reflects

the

inability

of

keratanase

to

digest equally

all

forms

of KS.

Table

2.

Generalized patterns

of

PG distribution

during neural

crest

development

Pattern

Antigens

Antibodies

I

II

III

rv

*

Following

CS-PG

Ch6S-PG

KS-PG

ChO-PG

KS-PG

CS-PG

Ch4S-PG

DS-PG

KS-PG

HS-PG

chondroitinase

AC II

7D4,

6C3, 4C3

3B3

5D4

1B5

4D1,

1B4, 2D3, 3D2

4D3

2B6*

2B6

8C2

33-2

digestion.

membrane, surrounding

the

neural tube

and

noto-

chord,

and in the

ventral portion

of the

sclerotome

(Fig. 2B). Fibrillar staining

was

also observed

in the

intersomitic clefts

and in the

perinotochordal region.

However, these

PGs

were generally absent from

regions through which neural crest cells initially will

migrate

(Fig.

3C,D; Bronner-Fraser, 1986b; Newgreen

et

al.

1986).

During

the

course

of

neural crest cell migration,

pattern

I

CS-PGs increased considerably

in

extent

and

588

R. Perris and others

intensity within the sclerotome (Fig. 3A,B)- Their

deposition appeared to follow a rostral-to-caudal and

ventral-to-dorsal sequence, first appearing in the

ventral sclerotome concomitant with sclerotome forma-

tion and later appearing in the dorsal sclerotome (4-5

somites rostrad). Twelve to fourteen somites rostral to

the most recently formed somite, pattern I CS-PGs

were observed throughout the sclerotome. The relative

levels of immunoreactivity within the different portions

of the sclerotome were quantitated by confocal laser

microscopy in conjunction with computerized image

analysis. The dorsal sclerotome, containing migrating

neural crest cells (Fig. 2F,G) exhibited a 10-fold lower

staining intensity for 7D4-immunoreactive CS-PGs