STEdev89.pdf

Development 107, 309-319 (1989)

Printed in Great Britain © The Company of Biologists Limited 1989

309

Jl/tenascin-related molecules are not responsible for the segmented

pattern of neural crest cells or motor axons in the chick embryo

CLAUDIO D. STERN

1

, WENDIE E. NORRIS

1

, MARIANNE BRONNER-FRASER

2

,

GEOFFREY J. CARLSON

1

, ANDREAS FAISSNER

3

, ROGER J. KEYNES

4

and MELITTA SCHACHNER

3

department of Human Anatomy, South Parks

Road,

Oxford 0X1 3QX, UK

2

Developmental Biology Center, University of California, Irvine, Ca. 92717, USA

^Department of Neurobiology, University of Heidelberg, 69, Heidelberg, West Germany

4

Department of Anatomy, Downing Street, Cambridge CB2 3 DY, UK

Summary

It has been suggested that substrate adhesion molecules

of the tenascin family may be responsible for the

segmented outgrowth of motor axons and neural crest

cells during formation of the peripheral nervous system.

We have used two monoclonal antibodies (M1B4 and

578) and an antiserum [KAF9(1)] to study the expression

of Jl/tenascin-related molecules within the somites of

the chick embryo. Neural crest cells were identified with

monoclonal antibodies HNK-1 and 20B4. Young somites

are surrounded by Jl/tenascin immunoreactive ma-

terial, while old sclerotomes are immunoreactive pre-

dominantly in their rostral halves, as described by other

authors (Tan

et

al.

1987 -

Proc. natn.

Acad.

Sci. U.S.A.

84,

7977; Mackie

et

al.

1988 -

Development

102,

237). At

intermediate stages of development, however, immuno-

reactivity is found mainly in the

caudal

half of each

sclerotome. After ablation of the neural crest, the

pattern of immunoreactivity is no longer localised to the

rostral halves of the older, neural-crest-free sclerotomes.

SDS-polyacrylamide gel electrophoresis of affinity-puri-

fied somite tissue, extracted using M1B4 antibody,

shows a characteristic set of bands, including one of

about 230xlO

3

, as described for cytotactin, Jl-200/220

and the monomeric form of tenascin. Affinity-purified

somite material obtained from neural-crest-ablated

somites reveals some of the bands seen in older control

embryos, but the high molecular weight components

(120-230X10

3

) are missing. Young epithelial somites

also lack the higher molecular mass components. The

neural crest may therefore participate in the expression

of Jl/tenascin-related molecules in the chick embryo.

These results suggest that these molecules are not

directly responsible for the segmented outgrowth of

precursors of the peripheral nervous system.

Key words: tenascin, cytotactin, Jl, segmentation, somites,

neural crest, motor axons, peripheral nervous system,

substrate-adhesion molecules (SAMs).

Introduction

The peripheral nervous system of higher vertebrate

embryos becomes segmented because neural crest cells

and motor axons emerging from the developing spinal

cord are restricted in their migration to the rostral half

of each adjacent sclerotome (Keynes and Stern, 1984;

reviewed by Keynes and Stern, 1988). The study of the

interactions between precursors of the peripheral ner-

vous system and somite tissue therefore constitutes an

apparently simple model system for the investigation of

factors controlling patterning in the developing nervous

system. Recently, our laboratories and others have set

out to find macromolecules that are differentially ex-

pressed in the two halves of the sclerotome, with the

aim of identifying those that have a function in control-

ling the segmental pattern (Stern

et al.

1986; Tosney

et

al.

1987; Tan

et al.

1987; Mackie

et al.

1988; Layer

et al.

1988;

Norris

et al.

1989; Halfter

et al.

1989).

One recent paper (Tan

et al.

1987) reported that the

glycoprotein cytotactin (main M

r

= 190-220xlO

3

; Gru-

met

et al.

1985; Hoffman

et al.

1988) is restricted to the

rostral halves of the sclerotomes of the chick embryo,

while a cytotactin-binding proteoglycan (CTB-proteo-

glycan; Hoffman and Edelman, 1987) is restricted to the

caudal halves. Tan

etal.

(1987) suggested that cytotactin

and its proteoglycan ligand

'may control the pattern of

neural

crest

migration'.

However, several of their results

310

C. D. Stern and others

suggest that the relationship between neural crest

migration and the expression of these macromolecules

is not straightforward. First, cytotactin is not localised

to the rostral halves of the sclerotomes present before

stage 16 (Hamburger and Hamilton, 1951) or so, at

which time the neural crest has already entered the

rostral halves of these sclerotomes (Thiery

et al.

1982;

Rickmann

etal.

1985; Bronner-Fraser, 1986; Loring and

Erickson, 1987; Teillet

et al.

1987. cf. Crossin

et al.

1986);

second, paradoxically, cytotactin substrates

in-

hibit

neural crest migration and spreading

in vitro;

third, it seems curious that while cytotactin is restricted

to the rostral half of the sclerotome, its proteoglycan

ligand becomes localised to the opposite half after the

appearance of cytotactin.

Comparable results were obtained by another group

of workers (Mackie

et al.

1988), who investigated the

distribution of tenascin (Chiquet and Fambrough, 1984;

Mackie

et al.

1987), a hexamer of glycoproteins of

similar molecular mass to cytotactin (Vaughan

et al.

1987),

in the sclerotome of the quail embryo. They, too,

found that tenascin is restricted to the rostral halves of

the sclerotomes after stage 16 (Mackie

etal.

1988), and

that tenascin substrates inhibit neural crest migration

and spreading

in vitro

when cultured on basal laminae

(Mackie

et al.

1988; Halfter

et al.

1989).

It seems likely that cytotactin and tenascin, as well as

two other glycoproteins, Jl 200/220 (Kruse

et al.

1985;

Faissner

et al.

1988) and GMEM (glioma extracellular

matrix molecule; Bourdon

etal.

1985) are related, if not

identical (see Aufderheide and Ekblom, 1988; Faissner

et al.

1988; Friedlander

et al.

1988; Hoffman

et al.

1988;

Mackie

et al.

1988; Halfter

et al.

1989). Their relative

molecular masses appear to be the same; all of them

comprise several polypeptides of various relative mol-

ecular masses between 110 and 260 X10

3

, with a pre-

dominant band at M

r

= 220xl0

3

, and both cytotactin

and tenascin are assembled as a hexamer, called a

'hexabrachiori

(Friedlander

et al.

1988). Tenascin and

Jl have been reported to be related immunologically,

and cytotactin and tenascin show apparently identical

patterns of immunohistological localisation in various

tissues (see Mackie

et al.

1988), as well as identical

effects on neural crest cells

in vitro.

At least when

isolated from brain, cytotactin and its proteoglycan

ligand can carry the HNK-1/L2 carbohydrate epitope

(Hoffman and Edelman, 1987; Kunemund

et al.

1988;

Friedlander

et al.

1988; Hoffman

et al.

1988), as does Jl

(Kruse

et al.

1985; Faissner

et al.

1988).

In this study, we have used three different antibodies,

two monoclonals (M1B4 - Chiquet and Fambrough,

1984 and MAb578 - Faissner

et al.

1988 and in prep-

aration) and one antiserum (KAF9(1); Faissner

et al.

1988 and in preparation), to study the distribution of

Jl/tenascin-related molecules in the sclerotome of the

chick embryo in more detail. We performed neural

crest ablations to examine the relationship between the

position of neural crest cells and the expression of these

molecules, and constructed an M1B4 affinity column to

purify the antigens for subsequent identification by

SDS-polyacrylamide gel electrophoresis.

Materials and methods

Embryos and operations

Fertile hens' eggs (Rhode Island Red hybrids) were incubated

at

38

°C for 2-3 days until they had reached Hamburger and

Hamilton (1951) stages 10-20. Embryos destined for ablation

of the neural crest were treated as follows: at about stage

10-12,

a l-5xl-5cm window was cut in the egg shell with a

scalpel, the embryo floated up to the level of the shell with

calcium- and magnesium-free Tyrode's saline (CMF), and

about 50^1 of a 1:10 solution of Indian ink (Pelikan Fount

India) in CMF injected into the subblastodermic cavity, to

improve contrast between the embryo and the underlying

yolk. Removal of the neural crest was then performed, under

a drop of CMF, using a microsurgical knife (Week, 15° angle),

Dewecker's scissors and a steel needle made from a N°l

entomological pin. The dorsal half of the neural tube spanning

the region between the caudal end of the segmental plate and

the 4th most recently formed somite (corresponding to some

14 segments in length) was excised. The ventral half of the

tube and the underlying notochord were left in place. After

the operation, the CMF was removed from above the embryo,

and about 2 ml thin albumen withdrawn with a syringe from a

hole in the blunt end of the egg; this lowered the embryo back

into the shell. The shell was then sealed with PVC tape and

the egg incubated at 38°C for a further 36-48 h, by which time

the embryos had reached stages 17-22. Three additional

embryos were incubated for a longer period, until they had

reached stages 25-27.

Control embryos were treated in an identical manner: the

vitelline membrane was cut above the embryo and, in most

cases,

deep cuts were made on either side of the neural tube.

After these manipulations, the embryos were lowered into the

shell, the eggs sealed and incubated exactly as for operated

embryos. All of the 107 operated embryos and 116 controls

survived the subsequent period of incubation.

Immunohistochemistry

Control and operated embryos destined for immunohisto-

chemistry were fixed in 100% ethanol for lh or in buffered

formol saline (4 % formaldehyde in phosphate-buffered saline

[PBS]) for 30 min, and washed well with PBS. They were then

placed in a 5 % solution of sucrose in PBS for 1-3 h, then in

20%

sucrose/PBS for

8-24h,

and finally infiltrated for 2-6h

at 38°C with a solution containing 7-5% gelatin (Sigma, 300

Bloom) and 15 % sucrose in PBS. They were then brought to

room temperature, to allow the blocks to set. These blocks

were stored at 4°C for no longer than a week prior to serial

sectioning (5-10

pun)

at -20°C in a Bright cryostat. The

sections were collected on gelatinised glass slides, air dried

and stored at 4°C until required.

Prior to immunohistochemical staining, the sections were

rehydrated and the gelatin removed by placing them in PBS at

38°C for a few minutes, then washed extensively with PBS.

They were then blocked with 3% bovine serum albumin

(BSA, Sigma) for 20 min, placed in the appropriate antibody

(see below) for lh at room temperature, washed extensively

with PBS, placed in the appropriate labelled secondary

antibody for 1 h at room temperature, washed again, and then

processed for immunofluorescence or immunoperoxidase.

For immunofluorescence, the slides were mounted in a

nonquenching medium (14% Gelvatol 20/30 [Fisons] con-

taining 8'5ragmr' diazobicyclo-octane [DABCO, Aldrich,

as anti-quenching agent], 30% glycerol and 350

/.tg

ml"

1

sodium azide as preservative in PBS, pH6-8), and viewed and

photographed under epifluorescence optics in an Olympus

JlItenascin in neural crest and somites

311

Vanox-T microscope, using supplementary exciter and barrier

filters to minimise spill-over between fluorescein and rhoda-

mine wavelengths. The supplementary filter combinations

used were: excitation EY455/emission B460 for fluorescein

(blue excitation, green pass) and excitation EO530/emission

O590 for rhodamine (green excitation, red pass). Lack of

spill-over may be confirmed by the reader by viewing the

colour figures with red/green glasses.

For immunoperoxidase, the slides were washed briefly in

O-lM-Tris-HCl and then incubated in a 500jUgml~' solution

of diaminobenzidine (Aldrich), to which H

2

C>2 was added to a

final concentration of 0-3 %. The slides were incubated in this

for about lOmin at room temperature, then washed in

running tap water for 30min, rinsed in distilled water and

mounted in Aquamount medium (BDH).

Antibodies

Neural crest cells present within the sclerotome of chick and

quail embryos display the HNK-1/L2 epitope, and therefore

monoclonal antibodies specific for this epitope (such as NC-1

or

HNK-1;

Tucker

et al.

1984) are often used to detect the

presence of neural crest cells in histological sections. Bronner-

Fraser (1986) and Teillet

et al.

(1987) have investigated the

expression of the HNK-1 epitope in neural crest cells in the

chick embryo in some detail, conclude that almost all the crest

cells that traverse the sclerotome express HNK-1 immuno-

reactivity (unlike some crest cells in the head and some that

take the dorsolateral pathway between the ectoderm and the

somite); HNK-1 is therefore a good marker for neural crest

cells present in the sclerotome at this stage of development.

Since Jl/tenascin-related molecules can carry the HNK-1/L2

epitope, we have used antibody 20B4 (which recognises a

different, as yet uncharacterised, epitope expressed by neural

crest cells; Dr J. Denburg, unpublished data) in addition to

HNK-1 to detect neural crest cells and to control for the

possibility of cross-reaction between the HNK-1 antibody and

Jl/tenascin-related molecules.

The primary antibodies used (dilutions in 0-3 % BSA/PBS)

were:

M1B4 supernatant

(Mouse igG, originally raised by Dr

Douglas Fambrough and obtained from the Developmental

Studies Hybridoma Bank). This was used at concentrations

between 1 and lO^gm!" (between 1:10 dilution and undi-

luted).

The antibody recognises the original 'myotendinous

antigen' (= tenascin = hexabrachion) described by Chiquet

and Fambrough (1984; see also Friedlander

et al.

1988) and an

epitope near the carboxy-terminal end of chain I of the

cytotactin molecule (Friedlander

et al.

1988).

578 purified

ascites

(Rat IgG monoclonal against Jl 200/220;

Faissner

et al.

1988 and in preparation). Used at 1:100

1

(g

KAF9(J)

(IgG fraction of rabbit antiserum directed against

mouse Jl 200/220-tenascin; Faissner

et al.

1988 and in

preparation). Used at 1:200 (25/*gml"').

HNK-1 supernatant

(Mouse IgM, originally raised by Abo and

Balch, 1981). Used 1:2 or undiluted (about 50/igml"

1

). The

neural crest specificity of this antibody is discussed by various

authors (e.g. Tucker

et al.

1984; Rickmann

et al.

1985;

Bronner-Fraser, 1986; Teillet

et al.

1987; Canning and Stern,

1988).

20B4 supernatant

(Mouse IgG, originally raised by Dr Jeff

Denburg, to chick dorsal root ganglion cells and obtained

from the Developmental Studies Hybridoma Bank). Used

undiluted (20^gmr').

In double-immunofluorescence studies, it was important to

use secondary antibodies that did not cross-react across

species or immunoglobulin types. After extensive tests to

ascertain this, the following antibodies were selected:

For mouse IgG:

(1) affinity-purified tetramethylrhodamine

isothiocyanate (TRITC)-labelled goat anti-mouse IgG (Fc

fragment specific; Sigma), used at 1:50 or (2) IgG fraction of

affinity-purified fluorescein isothiocyanate (FITC)-labelled

goat anti-mouse IgG (Fc fragment, y-chain specific; Cappel).

Used at 1:50 or

1:100.

For mouse IgM:

(1) affinity-purified FITC-labelled goat anti-

mouse IgM (ju-chain specific; Sigma), used at

1:100,

or (2)

TRITC-labelled sheep anti-mouse IgM (Fc fragment specific;

Serotec), used at 1:40.

For rat IgG:

affinity-purified TRITC- or FITC-labelled goat

anti-rat IgG (Fc fragment specific, tested for the absence of

cross-reactivity to mouse IgG by the manufacturers; Cappel

1213-1721). We confirmed the absence of cross-reactivity

between this antibody and mouse IgG ourselves in sections

that had been incubated with mouse monoclonals M1B4 or

20B4.

Used at a dilution of 1:100 (30^gml~').

For rabbit IgG:

affinity-purified TRITC- or FITC-labelled

goat anti-rabbit IgG (Fey chain specific; Cappel), used at 1:50

(20/igmr

1

).

To absorb out any remaining nonspecific binding, all

secondary antibodies were made up in 0-3% BSA in PBS

containing 1-5% normal goat serum, and the final working

solution absorbed against either fresh or formalin-fixed 2-day

chick embryo tissues for 30min under gentle agitation at room

temperature; after this, the working solution was centrifuged

for 3min at lOOg in a Micro-Centaur centrifuge. These

procedures were necessary to remove components of the

secondary antisera that were found to bind to various chick

tissues. In double-immunofluorescence experiments, the two

primary antibodies were added together in 0-3 % BSA/PBS;

the two secondary antibodies were also used in a single

solution.

In each experiment, the entire control or neural-crest-

ablated embryo (i.e. all the serial sections) was stained with

one combination of antibodies. At least two control and two

operated embryos were studied in this way for each antibody

combination. Table 1 shows the combinations of primary

antibodies used in single- and double-immunofluorescence

experiments.

Control sections were included in each experiment; in

these, incubation in 3 % BSA/PBS replaced the incubation in

primary antibody.

Electrophoretic characterisation of antigens

We were not able to detect antigens recognised by antibodies

M1B4, 578 or KAF9(1) in immunoblots made from polyacryl-

amide gels of unfractionated somite tissue under either

reducing or nonreducing conditions. For this reason, we had

to resort to affinity-extraction of the antigens from these

tissues. 45

/ig

of M1B4 IgG were purified by affinity from

culture supernatant using goat anti-mouse IgG agarose

(Sigma), following the manufacturer's protocol. The bound

IgG was eluted using 0-1 M-glycine and 045 M-NaCl (pH2-4),

Table 1.

Combinations of antibodies used for

immunofluorescence studies

M1B4

578

KAF9(1)

HNK-1

20B4

Total = 59

M1B4

4

2

3

18

-

578

2

4

2

5

2

KAF9(1)

3

2

4

5

2

HNK-1

18

5

-

4

20B4

_

2

4

312

C. D. Stern and others

and the fractions collected were neutralised with a small

volume of lM-Trizma base (Sigma). This purified IgG was

coupled to 0-5 ml CNBr-activated Sepharose-4B (Sigma)

following the manufacturer's instructions.

Strips of somite tissue (with some adherent ectoderm and

endoderm) were dissected from unoperated embryos or from

embryos from which the neural crest had been ablated

36-48 h earlier (as described above). Equivalent regions were

taken from both operated and unoperated embryos, ignoring

the first and last three pairs of somites opposite the operated

region; both control and operated embryos were at stages

18-22.

Two types of sample were dissected from younger

embryos. In one, the last four pairs of somites plus some

adherent tissues were dissected from embryos at stages 12-14

and treated identically to the other samples. In the other, the

same region was dissected but the neural tube and notochord

were included in the sample. In all cases, tissues were

collected in PBS containing a cocktail of protease inhibi-

tors:

lmM-phenymethylsulphonylfluoride (PMSF; Sigma),

lmM-N-ethyl maleimide (NEM; Sigma), lO^gml"

1

soybean

trypsin/chymotrypsin inhibitor (STI; Sigma), lmM-EDTA

and 1 mM-EGTA, and quickly frozen on solid CO2.

The frozen tissue samples were thawed and homogenised in

200 ,ul solubilization buffer, made up of 0-25 % sodium deoxy-

cholate, lOmM-Tris, 150mM-NaCl (pH8-3) containing the

cocktail of protease inhibitors described above. They were

then centrifuged at 11600g for 5min and the supernatants

collected for loading onto the affinity column. Samples were

incubated in the column for 45 min at room temperature, after

which the column was washed with 15 column volumes of

solubilisation buffer containing 300mM-NaCl. Elution was

then performed using a buffer containing 0-1 M-diethylamine,

150mM-NaCl and 0-25% sodium deoxycholate (pHll-5).

100

iA

fractions were collected and a protein assay performed

as a dot blot on Immobilon membrane (Millipore) using

AuroDye (Janssen). Protein-containing fractions were pooled

and concentrated by pressure dialysis against 0-1% deoxy-

cholate, 20mM-Tris, 150mM-NaCl, 1 mM-EGTA and lmM-

EDTA(pH8-3).

Approximately equal amounts of protein were loaded per

track of 4-15% SDS-polyacrylamide gradient minigels

(9 cm x 9 cm x 0-5 mm) using the Laemmli (1970) buffer sys-

tem in an apparatus designed as described by Matsudaira and

Burgess (1978). Electrophoresis was performed at 150 V for

90 min, or until the tracking dye was 1-5 cm above the bottom

of the gel. The resulting gels were silver-stained as described

previously (Canning and Stern, 1988; Norris

et al.

1989).

14

C-labelled, colour-coded molecular weight standards

(Amersham, Rainbow Markers) were run in one or two tracks

in each gel. A track containing a sample of purified mouse

Jl/tenascin was also included in each gel for comparison. This

preparation of Jl/tenascin was prepared by affinity purifi-

cation as described previously (Faissner

et al.

1988), using

postnatal mouse brain tissue as starting material. The eluted

fractions were dialysed against water and lyophilised.

Immunoblots of purified mouse brain tenascin were also

made. Each blot included radioactive relative molecular mass

markers and samples of purified Jl/tenascin, under reduced

and non-reduced conditions. Transfer to Immobilon mem-

branes was performed as described previously (Primmett

et al.

1989).

Each membrane was probed with one of the antibodies

used in this study, namely M1B4, 578 or KAF9(1), and

detected with either rabbit anti-rat followed by gold-labelled

goat anti-rabbit Ig for MAb578, or gold-labelled goat anti-

rabbit Ig for KAF9(1), or gold-labelled goat anti-mouse IgG

for M1B4. All gold-labelled antibodies were purchased from

Janssen (AuroProbe system).

Results

Immunohistochemistry

Identification of neural crest cells with antibodies

20B4 and HNK-1

Monoclonal antibody 20B4 is reported to be specific for

dorsal root ganglion cells and migrating neural crest

cells;

it also detects an antigenic determinant in the

ectoderm of chick embryos (Dr J. Denburg; data from

sheet obtained with supernatant supplied from the

Developmental Studies Hybridoma Bank). In our

hands,

20B4 was not as useful as HNK-1 in revealing the

distribution of neural crest cells in the sclerotome

because 20B4 staining was always of lower intensity and

it only recognised a subset of cells stained by

HNK-1.

Nevertheless, the overall pattern of staining seen with

this antibody in embryo sections was similar to that seen

with

HNK-1.

Jl I

tenascin-immunoreactivity

Most of the immunocytochemical procedures involved

double staining, using one antibody directed to Jl/

tenascin-related molecules and one directed to neural

crest cells. The combinations of antibodies used in these

experiments are summarised in Table 1. Identical re-

sults were obtained using all three antibodies directed

against Jl/tenascin [M1B4, 578 and KAF9(1)]. This

was confirmed in double-staining experiments combin-

ing the three antibodies, one pair at a time: in every

case,

the staining patterns in the rhodamine and fluor-

escein wavelengths were identical.

In all somites of the youngest embryos (stage-13 or

earlier) and in the most caudal 2-3 pairs of (epithelial)

somites of older embryos (stage 16-22), staining was

restricted to the extracellular matrix surrounding each

somite and to the matrix associated with mesenchymal

cells within the lumen of the epithelial somites, as

described by Crossin

et al.

(1986), Tan

et al.

(1987) and

Mackie

et al.

(1988). Also in accordance with Tan

et al.

(1987) and Mackie

et al.

(1988), staining within the

sclerotomes of the most cranial (older) somites of

embryos older than stage 16 was restricted to the rostral

half of each sclerotome; most of the cells expressing

Jl/tenascin immunoreactivity were not

HNK-1-

or

20B4-positive (Fig. 1 E, F). In addition, Jl/tenascin-

immunoreactive filamentous material was seen close to

the border between adjacent sclerotomes; this material

appeared to be associated with the intersegmental

vessels (Fig. 1 B). Immunoreactive extracellular ma-

terial was also seen between the dermomyotome and

the ectoderm at all segmental levels. Strong staining

was also seen between adjacent dermomyotomes, es-

pecially at the caudal margin of each (Fig. 1 A).

The pattern of staining seen in younger sclerotomes

(the 4-6 most caudal sclerotomes, between 4 and 10

segments rostral to the segmental plate) of embryos

beyond stage 13 was different from that described by

Tan

et al.

(1987) and by Mackie

et al.

(1988). In these

young somites, which have already begun to be col-

onised by migrating neural crest cells (Rickmann

et al.

Fig.

1.

Changes

in the

expression

of

Jl/tenascin-related molecules during development

of the

sclerotome.

(A)

Coronal

section through

the two

most caudal sclerotomes

of a

stage-14 embryo, stained with

M1B4 (red) and HNK-1

(green). Note

the intensely HNK-1-positive perinotochordal matrix,

and the

HNK-1-positive neural crest cells

in the

rostral (left) half

of

the sclerotomes. MlB4-positive material,

on the

other hand,

is

more intense

in the

caudal than

the

rostral halves.

(B) Sagittal section through

two

caudal sclerotomes

of a

stage-15 embryo, stained with

HNK-1 (red) and

KAF9(1) (green).

More intense KAF9(1) staining

is

seen

in the

caudal (right) halves

of the

sclerotomes, while many HNK-1-positive neural

crest cells have already entered into

the

rostral halves. KAF9(1) staining

is

also seen surrounding each somite

and

near

the

intersegmental vessels.

(C)

Oblique transverse section through

a

stage-15 embryo, stained with KAF9(1) (green)

and HNK-1

(red),

about

9

segments cranial

to the

segmental plate.

The

section passes through

the

rostral half

of the

sclerotome

on the

left

and

through

the

adjacent caudal half

on the

right. Note

the

abundance

of

KAF9(l)-positive material between

the

dermomyotome

and the

ectoderm, between

the

neural tube

and the

sclerotome,

and

within both halves

of the

sclerotome.

KAF9(l)-positive material

is

more abundant

in the

caudal half

at

this level. HNK-1-positive cells have entered deep into

the

rostral half

of the

sclerotome.

(D)

Coronal section through

two

more rostral sclerotomes (about

12

segments rostral

to the

segmental plate)

of a

stage-15 embryo, stained with

HNK-1 (red) and

MAb578 (green). MAb578-immunoreactivity begins

to

predominate

in the

rostral

half. (E)

Coronal section through three more rostral sclerotomes (about

15

segments rostral

to

the plate)

of a

stage-17 embryo, stained with

M1B4 (red) and HNK-1

(green). Within

the

sclerotome,

M1B4

immunoreactivity

is now

confined

to the

rostral (left)

half,

except

in the

most ventromedial portion

of the

sclerotome,

adjacent

to the

notochord.

(F)

Higher power view

of one of the

sclerotomes shown

in (E). HNK-1

(green)

and M1B4 (red)

immunoreactivity

do not

coincide except

in

very

few

areas.

In

each figure,

the

head

of the

embryo lies towards

the

left

of

the photograph. Scale bars:

50^m

(A-E),

25^m (F). d,

dermomyotome;

m,

myotome;

n,

notochord;

s,

sclerotome;

t,

neural tube;

v,

intersegmental vessel.

Fig. 2. Neural crest ablation and expression of Jl/tenascin-related molecules. (A) Low-power view of an oblique coronal

section through a stage-15 embryo after ablation of six segments worth of dorsal neural tube at stage 11, stained with HNK-1

and TRITC-labelled secondary antibody. Note the presence of neural crest cells in most of the sclerotomes in the operated

region. (B) Phase-contrast micrograph of a sagittal section through two neural-crest-free sclerotomes of a stage-16 neural-

crest-ablated embryo. Note that the rostral (left) halves display a reduction in cell number as compared with the

corresponding caudal halves. (C) Same section as in B above, stained with M1B4 (red) and HNK-1 (green). Note the

absence of HNK-1-positive cells, and the predominance of M1B4 immunoreactivity in the caudal half-sclerotome.

(D) Sagittal section through neural crest-free older sclerotomes (about 12 segments rostral to the segmental plate) of a stage-

17 embryo, stained with HNK-1 (red) and KAF9(1). Note the predominance of KAF9(1) immunoreactivity in the caudal

halves of the sclerotomes. [Compare the pattern of staining with that shown in Fig. 1E,F, which passes through the same

level of a similarly-staged but unoperated embryo]. (E) Sagittal section through neural-crest-free sclerotomes of a stage-17

embryo stained with HNK-1 (red) and KAF9(1) (green). In this embryo, both halves of the neural-crest-free sclerotomes

display KAF9(1) immunoreactivity. Scale bars: 100/im in A 50 ,um in B-E. The head of the embryo lies towards the left of

each photograph, a, aorta; d, dermomyotome; n, notochord; t, neural tube.