/. Embryol. exp. Morph. Vol. 56, pp. 169-178, 1980
Printed in Great Britain © Company of Biologists Limited 1980
] 69
Localisation and characterization of
acid mucopolysaccharides in the early chick
blastoderm
ByCH. VANROELEN 12 , L. VAKAET 1 AND
L. ANDRIES 1
From the Laboratory of Anatomy and Embryology,
R.U.C.A., Antwerp
SUMMARY
Acid mucopolysaccharides in the extracellular compartment of early chick blastoderms
(16 h of incubation) were labelled with tritiated glucosamine and/or [35S]sulphate. The
incorporation pattern was studied autoradiographically. Treatment with testicular hyaluronidase revealed a testicular hyaluronidase-sensitive fraction, mainly at the periphery of Middle
Layer and Deep Layer cells, and a testicular hyaluronidase-resistant fraction, mainly at the
ventral side of the Upper Layer.
A biochemical analysis, utilizing chondroitinase ABC and nitrous acid, followed by cellulose acetate electrophoresis, demonstrated the synthesis of a non-sulphated fraction, i.e.
hyaluronic acid and/or chondroitin, and a sulphated fraction, comprising two undersulphated
components, i.e. chondroitin sulphate, and heparan sulphate or heparin. The appearance of
different AMPS in specific areas of the early chick blastoderm is regarded as an early specialization of the extracellular compartment.
INTRODUCTION
It is generally accepted that the extracellular matrix plays an important role
in cellular interactions, necessary for normal morphogenesis (Bernfield &
Wessels, 1970). The presence or the synthesis of acid mucopolysaccharides
(AMPS) in the extracellular matrix has been demonstrated in the chick embryo
at different stages of development, and their correlation with cellular interactions has been suggested (Franco-Browder, De Rydt & Dorfman, 1963;
Kvist & Finnegan, 1970; Manasek, 1970; Manasek et al 1973; O'Hare, 1973;
Abrahamsohn, Lash, Kosher & Minor, 1975; Pratt, Larsen & Johnston, 1975;
Solursh, 1976). In this study, the extracellular matrix of the early chick blastoderm, prior to the establishment of recognizable cartilaginous material, was
examined for AMPS, using autoradiography and cellulose acetate electrophoresis. We wanted to investigate whether the localization of the labelled products
and their nature, could give some insight into the significance of these products.
1
Authors' address: Laboratory of Anatomy and Embryology, Rijksuniversitair Centrum,
Groenenborgerlaan 171, 2020-Antwerpen, Belgium.
2
Author to whom reprint requests should be sent.
170
CH. VANROELEN, L. VAKAET AND L. ANDRIES
MATERIALS AND METHODS
White Leghorn eggs from commercial stock were incubated for 16 h at 38 °C
to yield blastoderms of stage 5-6 (Vakaet, 1970), corresponding to Hamburger
& Hamilton's stage 3 + (1951). They were explanted according to the procedure
of New (1955), and incubated at 38 °C for 30 min. Thereafter the blastoderms
were cultured in a medium, containing 0-5 ml egg white and 0-5 ml Ringer
solution with 3 mg agar, in the presence of 15 /tCi D-[6-3H]glucosamine hydrochloride (19Ci/mmol) or 70 /«Ci [35S]sulphate (230 Ci/mmol) (The Radiochemical Centre Ltd., Amersham) for 1 h. Only normally developed embryos
were used further.
The blastoderms destined for autoradiography were fixed overnight, at room
temperature, in 10% (w/v) aqueous formalin, containing 0-5% (w/v) cetylpyridinium chloride, 2 % (w/v) calcium chloride and an excess of calcium
carbonate. The tissues were dehydrated in a graded series of alcohol, cleared in
xylene, and embedded in paraffin wax. Sections, 8 fim thick, were deparaffinized in xylene, and hydrated. Sections of blastoderms cultured with tritiated
glucosamine, were treated with a 0-1 % ( w / v ) solution of ovine testicular hyaluronidase Type II (Sigma Chemical Co.) (365 NF units/mg) in 0-1 M phosphate
buffer (pH 5-5) at 38 °C for 5 h. Control incubations were performed on alternate sections in a buffer solution containing the enzyme, inactivated in a
boiling-water bath for 10 min. The sections were coated by dipping into Ilford
L4 Nuclear Emulsion, diluted 1/1 (v/v) with distilled water, at 45 °C. The slides
were exposed for 6 weeks at 4 °C, and processed further according to the method
of Caro, Van Tubergen & Kolb (1962). Control incubations without labelled
precursors, and incubations with the labelled precursors but cultured at 4 °C,
were carried out. A total of 33 blastoderms was studied.
The amount of labelled macromolecules in three groups of three blastoderms
and their fixing medium was measured by chromatographing (a) the in vacuo
concentrated fixing medium on a PD-2 prepacked column (Pharmacia, Uppsala,
Sweden), and (b) the extract of the formerly fixed blastoderms in 4 M guanidin
hydrochloride on an analogous column.
The AMPS were further identified using cellulose acetate electrophoresis
after culturing the blastoderms with the two precursors in the same medium,
and under the same conditions as mentioned above. For each test, five to ten
blastoderms were frozen, thawed, homogenized, and digested with papain
(Sigma Chemical Co.) according to the method of Antonopoulos, Gardell,
Szirmai & De Tyssonsk (1964). The digest was boiled, cooled and centrifuged.
The supernatant was dialysed for 2 days against deionized water at 4 °C, and
lyophilized. Aliquots of the lyophilizate were treated with either (1) nitrous
acid or (2) chondroitinase ABC (Sigma Chemical Co.), respectively according
to Dische & Borenfreund (1950), and Yamagata, Saito, Habuchi & Suzuki,
(1968). Undegraded AMPS were recovered from the crude extract using dialysis.
Acid mucopolysaccharides in early chick blastoderm
Fig. 3
111
Fig 2 D , E
Upper layer
Middle layer
>— /
' ^ "**'
Fig. 2 B, C
Deep layer
Fig. 2A
Fig. 1. Outline of a transverse section through the anterior part of the primitive
streak of a chick blastoderm at stage 6.
The samples were again lyophilized and resolubilized in a minimal volume.
The different AMPS present in the mixture were separated, using cellulose
acetate electrophoresis in 0-05 M lithium chloride in 001 M-HC1 (pH 2-1),
and in 0-05 M natrium barbital (pH 8-6). Each experiment was performed at
least three times. Reference samples of AMPS, i.e. hyaluronic acid, chondroitin
sulphate A, B and C (Sigma Chemical Co.), and heparin (Leo Pharmaceutische
Producten) were co-electrophoresed. The samples were exposed to a potential
gradient of 10 V/cm for 45 min. The cellulose acetate membranes were stained
according to Breen, Weinstein, Andersen & Vess (1970). The dried membrane
was cut in 2 mm strips and solubilized. The radioactivity in each strip was
counted in a liquid scintillation counter (Packard Tri-Carb 3380).
RESULTS
A utoradiography
The incorporation of precursors was examined during the period that active
ingression is occurring in the primitive streak. At this stage the regression of
Hensen's node has not yet started, and the anlage of the head process is not
yet present. The layers of the germ are indicated by Upper Layer (UL) (closest
to the vitelline membrane), Deep Layer (DL) (comprising the various generations of the endoblast: endophyll, primary DL or hypoblast and definitive DL
or endoblast) and Middle Layer (all cells lying between UL and DL). A schematic representation of a section through the anterior half of the primitive
streak is shown in Fig. 1.
After incorporation of tritiated glucosamine for 1 h, all autoradiographs show
a typical incorporation pattern of tritium-containing macromolecules (Fig. 2 A,
B, D). A high density of grains is present at the periphery of ML and DL
cells. Moreover, a sharp positivity is observed at the ventral side of the UL,
where electron microscope studies (Low, 1967) detect a basal lamina, and at the
apical side of the UL in the extraembryonic region. It is remarkable, that during
their ingression, the UL cells acquire the capacity to synthesize these molecules.
172
CH. VANROELEN, L. VAKAET AND L. ANDRIES
* . • • • .
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Acid mucopolysaccharides in early chick blastoderm
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.
173
,
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Fig. 3. Autoradiograph (focused on extracellular grains) of an early chick blastoderm
(St. 5-6), after [36S]sulphate incorporation. A section through the anterior half
of the primitive streak is shown (see Fig. 1). The ventral side of the UL is positive.
ML and DL show a diffuse label. Scale bar is 50 /tm.
Cellular grain positivity is observed over ML and DL cells, but is nearly absent
over the UL cells.
The positivity mentioned above, is largely removed after treatment with
testicular hyaluronidase, a hydrolytic enzyme degrading hyaluronic acid,
chondroitin sulphate A and C, and chondroitin (Fig. 2C, E). Thus, the positivity after tritiated glucosamine incorporation can probably be attributed to one
or more of these products. Residual positivity is most pronounced where a basal
lamina is present, indicating the synthesis of glycoproteins or testicular hyaluronidase-resistant AMPS (heparan sulphate or heparin, dermatan sulphate,
keratan sulphate). However, we cannot exclude the presence of incompletely
degraded hyaluronic acid or chondroitin sulphate A, C in a protecting matrix.
[35S]sulphate incorporation resulted in a different distribution of silver grains
FIGURE 2
Autoradiographs (focused on extracellular grains) of an early chick blastoderm
(St. 5-6), after [3H]glucosamine incorporation. Sections through the anterior half
of the primitive streak are shown.
(A) A general view. One half of the primitive streak and the lateral area of the
embryo. Scale bar is 100/*m.
(B) Central region (see Fig. 1) after treatment with a buffer solution containing
the heat-inactivated enzyme. Positivity is localized at the periphery of ingressing
UL cells, ML and DL cells. In the groove, some label is present at the dorsal side
of ingressing cells. Scale bar is 20 /on.
(C) Central region after testicular hyaluronidase pretreatment. A residual
positivity is observed in ingressing UL cells, ML and DL cells. Scale bar is 20 /*m.
(D) Lateral region (see Fig. 1) after treatment with a buffer solution containing
the heat-inactivated enzyme. The ventral side of the UL and the periphery of the
DL cells are positive. The apical surface of the UL is negative. Scale bar is 20 /tm.
(E) Lateral region after testicular hyaluronidase pretreatment. The ventral side of
the UL remains positive. The DL positivity is markedly reduced. Scale bar is 20 /*m.
12
EMB 56
174
CH. VANROELEN, L. VAKAET AND L. ANDRIES
(Fig. 3). The cells of the UL, ML and DL did not show any preference for
synthesis of sulphated products. In the extracellular compartment, an intense
label was observed at the ventral side of the UL. The sulphated products were
also present in the extracellular area of ML and DL.
Control experiments of blastoderms cultured (a) without the labelled precursors and (b) with the labelled precursors but at 4 °C, fail to show any positivity, indicating the absence of chemography or adsorptive effects during autoradiography.
Measurement of labelled macromolecules released in the fixing medium
The proportion of labelled macromolecules in the fixing medium, if compared
with the labelled macromolecules in the blastoderms, is, for tritiated macromolecules, certainly less than 10%, and for sulphated macromolecules certainly less than 5 %.
Cellulose acetate electrophoresis
The nature of the labelled products was further analysed using cellulose
acetate electrophoresis. According to Breen et al. (1970), the LiCl/HCl medium
allows a clear separation on the basis of the sulphate content/disaccharide
ratio. We confirmed this by means of migration of standard AMPS. The hyaluronic acid (non-sulphated) remains near the origin, while heparin, showing a
higher degree of sulphation than chondroitin sulphate A, B and C, migrates
faster than the latter products (Fig. 4B).
The sample, containing labelled AMPS from blastoderms (Fig. 4A), shows
three components: a tritium band near the origin, and two other bands, both
containing tritium as well as pSJsulphate. The tritiated band (peak 1 in
Fig. 4A) comigrates with reference hyaluronic acid. Although the synthesis of
this product is probable, the possibility of the synthesis of non-sulphated chondroitin cannot be ruled out. The two double-labelled bands (peak 2 and 3 in
Fig. 4 A) migrate more slowly than the reference chondroitin sulphates. Consequently, those products show a lower sulphate content/disaccharide ratio than
the reference chondroitin sulphates. This undersulphation does not seem to be
the result of the procedure, because it was also observed by other authors using
different techniques (Franco-Browder et al. 1973; Kvist & Finnegan, 1970;
Manasek et al. 1973).
It is difficult to characterize AMPS with cellulose acetate electrophoresis
alone, particularly since the components are undersulphated. Therefore, this
technique was combined with the pretreatment of the sample with (1) nitrous
acid, which degrades specifically JV-sulphated AMPS, i.e. heparin sulphate or
heparin (Inoue & Nagasawa, 1976), or (2) chondroitinase ABC, which degrades
chondroitin sulphate A, B and C. The slowest double-labelled band (peak 2
in Fig. 4A) is sensitive to nitrous acid, and the second (peak 3 in Fig. 4A)
to chondroitinase ABC.
Acid mucopolysaccharides in early chick blastoderm
175
12
(A)
11
V
o
10
(B)
20
30
mm migration
Hya. acid
10
40
CH. S A/B/C
•20
30
mm migration
40
50
Fig. 4. Cellulose acetate electrophoresis in 0.05 M-LiCl/0.01 M-HCI. Potential gradient 10 V/cm for 45 min. (A) Separation of [3H]glucosamine (
) and L35S]sulphate (
) labelled AMPS of the early chick blastoderm. Peak 1: (3 +1) mm; peak
2: (19 + 2) mm; peak 3: (27±2) mm. (B) A densitometer trace (629 nm) of a
mixture of commercial hyaluronic acid, chondroitin sulphate A, B and C, and
heparin.
Electrophoresis in Na-barbital gives a well-separated tritium band, coinciding
with hyaluronic acid. The quality of separation of the sulphated components
in the labelled sample was lower in the Na-barbital medium, compared with the
LiCl/HCl medium.
DISCUSSION
The use of autoradiography in the study of the localization of AMPS relies
on the efficient fixation of those macromolecules. Leaching of AMPS from cow
12-2
176
CH. VANROELEN, L. VAKAET AND L. ANDRIES
uterus and vagina into the fixative (formalin-cetyltrimethylammonium bromide)
did not appear to be more than 10 % (Radmehr & Butler, 1978). Because Pousty,
Bari-Kahan & Butler (1975) demonstrated that the solubility of AMPS in
fixatives varies with the tissue source, the amount of solubilized labelled macromolecules from the blastoderms into the fixative was measured. Our results
suggest that in chick blastoderms, the solubilization of AMPS into the fixative
is negligible.
Tritiated glucosamine incorporation was carried out in order to study the
extracellular AMPS. However, because glucosamine is a precursor of a wide
variety of biological molecules (Warren, 1970), the labelled material had to
be treated with testicular hyaluronidase, before processing for autoradiography.
Our results reveal that the positivity of ML and DL cells largely disappears after
this treatment. Because the testicular hyaluronidase preparations often contain
some proteolytic activity, the loss of radioactivity in the autoradiographs could
also be caused by the removal of glycoproteins from the sections, as tritiated
glucosamine will label glycoproteins as well as glycosaminoglycans. Preliminary
results of the separation of the labelled product in the papain digests, using gel
chromatography, reveal that the proportion of tritiated low-molecular-size
glycopeptides, if compared to the tritiated AMPS, is small. Therefore, synthesis
of hyaluronic acid, chondroitin and chondroitin sulphate A and/or C may be
assumed in ML and DL cells. Subsequently, evidence is provided for the
synthesis of hyaluronic acid, chondroitin, and/or chondroitin sulphate A, C.
Cinematographic observations (Vakaet, 1970) clearly demonstrate that ML and
DL cells are actively migrating at this stage of development. The synthesis of
this testicular hyaluronidase-sensitive fraction was also observed in the UL
cells during their ingression through the primitive streak, and their appearance
in the ML. These autoradiographic results allow us to propose a correlation
between the synthesis of testicular hyaluronidase-sensitive AMPS, and the
migration of individual cells in the early chick blastoderm. The possible importance of AMPS, as components necessary for normal morphogenesis during
this early stage of development, was also suggested by Solursh (1976). He proposed that the synthesis and secretion of hyaluronic acid, followed by extensive
hydration (Ogston & Stanier, 1951), could result in the expansion of forming
extracellular spaces. This cell-free space would then favour the migration of the
ML and DL cells.
The incorporation of psjsulphate into macromolecular material has been
used in this investigation as an assay for the synthesis of sulphated AMPS
(for review see Kosher & Searls, 1973). However, the direct sulphation of proteins (Slomiany & Meyer, 1972) cannot be eliminated. The ubiquitous distribution of cells in the early chick blastoderm, which share the ability to synthesize
these sulphated products, prove that this phenomenon is not a unique characteristic of cells undergoing chondrogenesis (for review see Abrahamsohn et al.
1975). It has also been suggested that all embryonic cells pass through a chondro-
Acid mucopolysaccharides in early chick blastoderm
111
genie phase (Holtzer & Matheson, 1970). However, in agreement with Kosher
& Searls (1973), we feel it is more reasonable to assume that the synthesis of
this material is necessary for normal morphogenesis during the earliest stages
of development. Moreover, O'Hare (1973) demonstrated histochemically that
qualitative as well as quantitative differences in AMPS occur with the onset
of chondrification. The undersulphation of the sulphated AMPS, detected in the
early chick blastoderm, could be one of these structural differences, and is currently under investigation in our laboratory.
The electrophoretical results clearly demonstrate the synthesis of non-sulphated and sulphated AMPS. Although the former contains the bulk of the
tritium label, no conclusions about the total amount, or the rate of synthesis
of AMPS can be made. In order to do this, knowledge of the specific activity
of the labelled products, and of the precursor pools is necessary, which is very
difficult because of the small amount of material available. In this study, we
have limited ourselves to a consideration of the AMPS in the extracellular
compartment. Other components of this compartment such as collagen (Johnson, Manasek, Vincon & Seyer, 1974), or glycoproteins (Manasek, 1975; Greenberg & Pratt, 1977; Mintz & Glaser, 1978) were not studied. The appearance of
different AMPS in specific areas of the early blastoderm suggests an early
specialization of the extracellular matrix.
We should like to thank Mrs V. Gonthier-Van der Stock for technical assistance and Mrs
N. Van den hende-Bol for typing the manuscript.
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{Received 20 March 1979, revised 1 October 1979)