/. Embryo!'. exp. Morph. Vol. 42, pp. 195-207, 1977
Printed in Great Britain © Company of Biologists Limited 1977
195
Glycosaminoglycan localization
and role in maintenance of tissue spaces
in the early chick embryo1
By MARILYN FISHER 2 AND MICHAEL SOLURSH 2
From the Department of Zoology, University of Iowa
SUMMARY
Comparison of sections stained with Alcian blue at pH 10 or 2-5 demonstrates the distribution of sulfated and non-sulfated glycosaminoglycans in the extracellular matrix of the
stage-8 (Hamburger & Hamilton, 1951) chick embryo. Both types of GAG are present in
basement membranes throughout the embryo. Treatment of sections with Streptomyces
hyaluronidase, reported to be specific for hyaluronic acid, prior to staining with Alcian
blue at pH 2-5 reveals that hyaluronate is an important constituent of the extracellular
matrix in basement membranes and in intercellular spaces within the mesoderm. Hyaluronate
is shown to be the predominant glycosaminoglycan in the matrix of the head mesenchyme.
In addition, examination by SEM and light microscopy of embryos after treatment in ovo
with hyaluronidase shows that removal of hyaluronate from living embryos results in a
dramatic decrease in cell-free spaces and a weakening of the association between mesoderm
and ectoderm in the head.
INTRODUCTION
It has become increasingly apparent during recent years that molecules
present in the matrix surrounding cells may have profound influences on the
morphogenetic activity and cytodifferentiation of those cells. Specific examples
include the influence of collagen on corneal differentiation (Dodson & Hay,
1973; Meier & Hay, 1974), the influence of glycosaminoglycans (GAG) on
organogenesis (Bernfield, Cohn & Banerjee, 1973), and the effect of notochordal collagen and GAG on somite chondrogenesis (Strudel, 1973; Kosher &
Lash, 1975). One such molecule, hyaluronic acid (HA), is a GAG commonly
found associated with extracellular matrix (ECM) in adult and embryonic
tissues (see Toole, 1976, for review). In chick embryos, for example, HA has
been shown to be present in the matrix of migrating sclerotome (Kvist &
Finnegan, 1970) and neural crest cells (Pratt, Larsen & Johnston, 1975) as
well as in the developing heart (Manasek et al. 1973) and limb (Toole & Trelstad,
1971; Toole, 1972). Furthermore, HA has been shown to be synthesized in
1
Part of this work appeared previously in abstract (Fisher and Solursh, 1976).
Authors' address: Department of Zoology, University of Iowa, Iowa City, Iowa 52242,
U.S.A.
2
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M. FISHER AND M. SOLURSH
high proportions relative to other GAG by gastrula-stage chick (Manasek,
1975; Solursh, 1976) and rat (Solursh & Morriss, 1977) embryos.
The work presented here demonstrates that HA is also a constituent of the
ECM of chick primary mesenchyme and that its continued presence is necessary
for maintenance of the extensive intercellular spaces which are characteristic
of the mesenchyme in the head region of the embryo. Three experimental
approaches have been used: (1) paraffin sections of normal embryos were
stained with Alcian blue at pH 1-0 or 2-5 to illustrate the distribution of sulfated
and non-sulfated GAG; (2) tissue sections were treated with specific hyaluronidases prior to Alcian blue staining to localize HA specifically; and (3) tissue
sections prepared from embryos incubated in ovo with hyaluronidase for 3 h
before fixation were examined for morphological abnormalities which might
result from disruption of the integrity of the ECM by HA removal.
MATERIALS AND METHODS
Incubation and enzyme treatment of embryos
Chick embryos [White Leghorn (Welp Inc., Bancroft, la.) or Hubbard
Golden Comet (Johnson Co. Feed & Hatchery, Iowa City, la.)] to be used
only for histochemical examination were incubated undisturbed to stage 8
(Hamburger & Hamilton, 1951). Embryos to receive in ovo enzyme treatment
were incubated and allowed to reach late head-process or early head-fold
stage (stage 5 or 6 respectively) and then candled to determine the position of
the embryo so that a hole could be cut in the shell directly over the embryo.
These embryos then received a sub-blastodisc injection of 20 fi\ of testicular
hyaluronidase, 400 u/ml (Worthington, HSEP), Streptomyces hyaluronidase,
200 u/ml (Calbiochem), or Howard Ringer. The eggs were then resealed with
Parafilm and returned to the incubator for an additional 3 h incubation.
Fixation of embryos for light and scanning electron microscopy
Embryos for scanning EM observation were fixed in 2 % glutaraldehyde
in saline G for 1-20 h and postfixed by treatment with 1% OsO4 in 0-1 M
cacodylate buffer at pH 7-4 and thiocarbohydrazide as described by Kelley,
Dekker & Bluemink (1973). The fixed specimens were then dried by the critical
point method, gold-coated and examined in a Cambridge S4 stereoscan microscope.
Some injected embryos and the control embryos which were to be prepared
for Alcian-blue staining and enzyme treatment of sectioned material were
fixed for 1 h in Carnoy's fixative containing 0-5 % cetylpyridinium chloride,
then dehydrated through an alcohol series and embedded in paraffin.
Glycosaminoglycan localization in early chick embryo
197
Enzyme treatment and Alcian blue staining of paraffin sections
Deparaffinized, hydrated sections were incubated for 2 h in the presence of
100 u/ml Streptomyces hyaluronidase, 400 u/ml testicular hyaluronidase,
or 0-1 M phosphate buffer, pH 5-0 (Yamada, 1971). Following enzyme treatment, sections were stained with 1 % Alcian blue at pH 2-5 or 1-0 (Humason,
1972), then dehydrated, cleared in xylene and mounted in permount.
Enzyme specificities
Streptomyces hyaluronidase was chosen because of its reported specificity
for hyaluronate (Ohya & Kaneko, 1970). Testicular hyaluronidase degrades
chondroitin and chondroitin sulfates A and C as well as hyaluronate. Both
enzymes were tested for possible contamination with protease (Davis & Smith,
1955) and no protease activity was found in either enzyme. In addition, it is
unlikely that the effects of hyaluronidase treatment reported here are due to
toxic effects of the digestion products. Hughes, Freeman & Fadem (1974)
studied the teratogenic effects of a number of sugars including mono-, di-, and
trisaccharides. While all sugars tested were found to be teratogenic, the abnormalities reported did not include those reported in this paper.
RESULTS
Distribution of GA G
When sections stained with Alcian blue at pH 2-5 (stains all GAG) are
examined, one sees in the head region that there is an extensive basement
membrane underlying the neural and non-neural ectoderm and surrounding the
notochord. The basement membrane appears to stain more intensely under the
neural ectoderm (particularly the lateral curved surfaces) and around the notochord than under the non-neural ectoderm (Fig. 1 A). Underlying the endoderm
in the region where the foregut is closed, intensely staining basement membrane is
associated only with the ventral endodermal wall (not shown). Posterior to the
anterior intestinal portal there is staining basement membrane associated with
the endoderm only in the region of the lateral body fold, where the splanchnic
mesoderm appears organized as a columnar epithelium. In addition to the
basement membrane, Alcian-blue-staining material is abundant in association
with the head mesenchyme. The material appears precipitated on the surfaces
of cells as small granules and as more extensive aggregates (Fig. 1D).
In sections stained at pH 1-0 (stains only sulfated GAG), the basement
membrane is still visible although reduced somewhat in staining intensity
(Fig. IB). This is particularly true of the endodermal basement membrane
which stains only as patches, if at all, in the ventral wall of the closed foregut
(not shown). The Alcian-blue-staining material present in association with the
head mesenchyme is virtually eliminated at this pH. It may be concluded from
198
M. FISHER AND M. SOLURSH
IB
Fig. 1. Sections through the head region of a stage-8 embryo. (A) Section stained
with Alcian blue at pH 2-5, showing intensely stained basement membrane (large
arrows) associated with neural ectoderm, spanchnic endoderm and, to a lesser
extent, the head ectoderm. Also visible is intensely staining intercellular matrix
material (small arrows) which here appears mainly precipitated on the surfaces of
mesodermal cells, although it also may appear more dispersed as granular material
suspended in the intercellular space, x 160. (B) Section stained with Alcian blue
at pH 10 to demonstrate the distribution of sulfated GAG in the extracellular
matrix. Note the reduced staining intensity of the basement membrane and more
strikingly of the intercellular matrix. At this pH, staining material is also visible
on the external surface of the ectoderm, particularly the neural ectoderm, x 160.
(C) Section incubated with Streptomyces hyaluronidase and stained with Alcian blue
at pH 2-5. Note the absence of heavily staining basement membranes and intercellular matrix, x 160. (D) High magnification micrograph of a region from (A)
next to the neural ectoderm which shows the darkly staining matrix material
associated with the surfaces of cell bodies and cell processes, x 1000.
Fig. 2. Section through the head region of a stage-8 embryo incubated live in the
presence of Streptomyces hyaluronidase. The section is stained with Alcian blue at
pH 2-5. Note the clumped appearance of the head mesenchyme, the absence of
stainable matrix material associated with the mesenchymal cells and basement
membrane, and the apparent loss of attachment between the mesenchyme and
both the neural and head ectoderm, x 160.
Glycosaminoglycan localization in early chick embryo
199
these results that there is a considerable contribution by both sulfated and nonsulfated GAG to the basement membranes in the head region, while material
associated with the mesenchyme is predominantly non-sulfated.
More posteriorly in the embryo, at the region where somites have formed,
staining at pH 2-5 reveals a prominent basement membrane underlying the
ectoderm (particularly the neural ectoderm and adjacent non-neural ectoderm
over the somites) and surrounding the notochord (Fig. 3 A). The endoderm,
again, appears to lack a prominently staining basement membrane. If present
at all, it seems to be confined to the region underlying the intermediate mesoderm. The somites themselves are seen to be surrounded by a basement membrane which contains Alcian-blue-staining material, as are the dorsal surface
and, to a lesser extent, the ventral surface of the lateral plate mesoderm.
Between the dorsal surface of the lateral plate mesoderm and the ectoderm, as
between the ventral surface and the endoderm, are seen heavily staining granules
associated with cell processes. Granules are increasingly abundant more
laterally. The lateral plate mesoderm cells at this level are quite closely packed
and there is no significant stainable material within the cell mass.
When the somite region is stained at pH 1-0, the same distribution of ECM
as seen at pH 2-5 is evident. Stainable material is present in basement membranes and as granules between the mesoderm and ectoderm or endoderm as
well as within the more disperse lateral mesoderm, but the staining intensity
is somewhat reduced (Fig. 3B). These results show that in this region the ECM
contains predominantly sulfated GAG.
In the yet unsegmented region between the somites and the primitive streak,
nearly the same distribution of Alcian-blue-staining ECM is seen as in the
somite region. Closer to the region of Hensen's node there is a progressive
change in the amount of staining matrix until, at the node, there is a slight
basement membrane underlying the lateral ectoderm only. This is visible when
stained at either pH (Fig. 4A, B). At this level there appears to be occasional
staining material associated with the mesoderm, particularly in the more
lateral portions. This material stains less intensely at pH 1-0. These results
indicate that in the axial region of the embryo from the level of the somites
through the primitive streak, Alcian-blue-staining ECM is predominantly
composed of sulfated GAG with a small contribution by non-sulfated GAG.
Non-sulfated GAG becomes increasingly evident in the more lateral extremities
of the embryo at this level.
Distribution of hyaluronate
The presence of HA is demonstrated by comparison of sections incubated in
buffer with and without Streptomyces or testicular hyaluronidase and then
stained with Alcian-blue at pH 2-5. Streptomyces hyaluronidase specifically
degrades HA while testicular hyaluronidase degrades chondroitin and chondroitin sulfates as well as hyaluronate, yet treatment with either enzyme yields
similar results.
200
M. FISHER AND M. SOLURSH
4A
4B
* , ^ L
Figs. 3 and 4.
Glycosaminoglycan localization in early chick embryo
201
In the head region all Alcian-blue-stainable material visible in buffer-treated
sections (as described above) appears to have been removed by enzyme treatment (Fig. 1C). Some residual patches of stainable basement membrane may be
seen associated with the neural fold ectoderm in enzyme-treated sections
stained at pH 1-0 (not shown). This is probably visible because of the reduced
intensity of background staining at this pH. The amount of stainable basementmembrane material seen under these conditions is slight compared to nonenzyme-treated sections stained at pH 1-0. These results show that HA is
present in the basement membrane as well as in the ECM associated with the
head mesenchyme. Furthermore, removal of hyaluronate by Streptomyces
hyaluronidase digestion results in loss of most sulfated GAG, which must be
intimately associated with HA in the ECM.
In the somite region, following enzyme treatment some stainable basementmembrane material is still present underlying the neural ectoderm and, to a
lesser extent, on the dorsal surfaces of the somite and lateral plate mesoderm.
FIGURES 3 AND 4
Fig. 3. Sections through somite region of the same embryo as in Fig. 1.
(A) Section stained with Alcian blue at pH 2-5 showing heavily staining basement
membranes underlying surface and neural ectoderm and surrounding the somite
and notochord. There is also heavily stained extracellular material associated
with the dorsal surface of the lateral plate mesoderm and, to a lesser extent, with
cell processes extending from the mesoderm to the ectoderm and endoderm. x 160.
Inset: high-magnification micrograph of a region between the mesoderm and
ectoderm which shows cell processes with associated Alcian-blue-staining granules.
x500.
(B) Section stained with Alcian blue at pH 10 to demonstrate the distribution of
sulfated GAG in the extracellular matrix. Note that the distribution of staining
material is the same as in (A), although here there is some reduction in staining
intensity. Again, some stainable material is also visible on the external surface of the
ectoderm when stained at this pH. x 160.
(C) Section incubated with Streptomyces hyaluronidase prior to staining with
Alcian blue at pH 2-5. Note absence of stainable matrix indicating an integral
contribution of hyaluronate to the extracellular matrix, x 160.
Fig. 4. Sections through Hensen's node region of the same embryo as in Figs. 1
and 3.
(A) Section stained with Alcian blue at pH 2-5 demonstrating the relative lack of
staining extracellular matrix material. Some basement membrane is visible underlying the lateral ectoderm; in addition some darkly staining material appears
associated with the ventral surface of the mesoderm. x 160.
(B) Section stained with Alcian blue at pH 10 to demonstrate the distribution of
sulfated GAG in the extracellular matrix. Note that there is a faint basement
membrane underlying the lateral ectoderm only. As in the anterior region, stainable
matrix is visible on the external surface of the ectoderm, x 160.
(C) Section incubated with Streptomyces hyaluronidase prior to staining with
Alcian blue at pH 2-5. The absence of stainable matrix indicates that hyaluronate
is an integral part of the small amount of extracellular material present in this
region, x 160.
202
M. FISHER AND M. SOLURSH
However, the granular material present associated with cell processes between
the mesoderm and ectoderm or endoderm is no longer visible (Fig. 3 C) nor is
the granular material associated with the more disperse lateral mesoderm. This
is true also of the ECM material in the unsegmented mesoderm posterior to the
somites after enzyme treatment, and is not unexpected in light of our previous
observations that most stainable material in these regions is sulfated GAG.
However, some HA is also present here, predominantly associated with the
more lateral mesoderm and the spaces separating the mesoderm from ectoderm
and endoderm.
Finally, in Hensen's node area, where very little was present initially, enzymetreated sections are virtually devoid of Alcian-blue-staining ECM. Faint traces
of basement membrane remain under the lateral ectoderm (Fig. 4C).
Effect of hyaluronidase treatment on living embryos
The above results demonstrate that HA is associated with the ECM in all
regions of the young embryo, and is particularly abundant in cell-free spaces
associated with mesoderm. More non-sulfated, Alcian-blue-staining material
sensitive to Streptomyces hyaluronidase is found in association with the head
mesenchyme than anywhere else in the embryo. In an attempt to understand
the developmental significance of this molecule, embryos were incubated
in ovo for 3 h after the injection of hyaluronidase and then fixed and examined
histologically to determine any morphological abnormalities which might
result from this treatment. Similar results are obtained when either Streptomyces
or testicular hyaluronidase is injected. The most striking result is the loss of
cell-free spaces associated with the mesoderm. This effect of HA removal is
most obvious in the head mesenchyme (Fig. 2). After enzyme treatment the
mesenchyme cells appear clumped and resting on the endoderm. The mesenchyme only rarely retains connection with either the neural or non-neural
ectoderm. This tissue separation is demonstrated consistently in both light
microscopic and SEM (Fig. 6) preparations of enzyme-treated embryos only,
and is therefore not attributable to an artifact of either Carnoy fixation or
paraffin embedding. The space under the neural groove, between the neural
ectoderm and the endoderm, which normally is at least two cell diameters in
width, is now reduced to as little as one third of its former size with barely
enough room for a single flattened cell, and space between mesenchymal cells
appears by light microscopy to be completely gone (Fig. 2). The notochord
is accordingly flattened and somewhat disorganized in appearance. In addition,
there is a conspicuous decrease in ECM which can be seen by SEM (Figs. 5-8) or
by Alcian-blue staining of sections from enzyme-treated embryos (Fig. 2). The
SEM micrographs show that there is ECM material present in control embryos
(Figs. 5, 7) which is absent from enzyme-treated embryos (Figs. 6, 8). The
reduction of Alcian-blue-stainable matrix is seen throughout most embryos.
In these embryos (Fig. 2), stainable matrix appears to be as completely removed
Glycosaminoglycan localization in early chick embryo
203
5"
Fig. 5. Scanning electron micrograph of the head region of a control embryo
(stage 8) showing the extensive network of fine cell processes (large arrows) and
strands (small arrow) of matrix material extending between the head mesenchyme
(HM) and neural (NE) and head ectoderm (HE), x 1600.
Fig. 6. Scanning electron micrograph of the head region of an embryo incubated
in ovo with Streptomyces hyaluronidase showing loss of extracellular matrix
material, and cell processes extending between the head mesenchyme and the
ectoderm. xl600.
204
M. FISHER AND M. SOLURSH
Fig. 7. Scanning electron micrograph which shows the extracellular material
associated with the basal surface of the head ectoderm of a stage-8 control embryo.
Note the fuzzy granular material (arrows) associated with the cells' basal surfaces
and with the strands hanging from the cells, x 3120.
Fig. 8. Scanning electron micrograph of the basal surface of the head ectoderm of a
stage-8 embryo which was incubated in ovo in the presence of Streptomyces hyaluronidase. Strands visible here appear mainly to be extremely fine filopodia and are not
associated with the fuzzy granular material seen in Fig. 7. Also note the sharp outlines of the cells indicating the loss of basement membrane material, x 5200.
Glycosaminoglycan localization in early chick embryo
205
as it is when sections are incubated with hyaluronidase. Some embryos appear to
have no significant reduction of Alcian-blue-staining matrix, but show clumped
mesenchyme. This probably reflects variation in the effective enzyme concentration in the embryo through inconsistency in making the injection, so that the
amount of enzyme present may be sufficient to alter the hydrostatic properties
of the matrix but not its stainability. These observations indicate that HA is
required for the maintenance of the extensive intercellular spaces characteristic
of head mesenchyme, and that the removal of HA from the ectodermal basement membrane results in a less stable interaction between mesenchyme cells
and the ectoderm as evidenced by the absence of connections between these two
tissues in sections from enzyme-treated embryos.
DISCUSSION
Solursh (1976) demonstrated that chick embryos at the stage studied here are
synthesizing GAG in the following relative proportions of HA/chondroitin
sulfate/heparan sulfate: 22/1-5/1. The results reported here provide histochemical evidence for the presence and location of both sulfated and nonsulfated GAG as constituents of the ECM throughout the early chick embryo.
Both types of GAG are associated with epithelial basement membranes at all
levels of the embryo to varying degrees, while hyaluronate predominates in
intercellular spaces. In these young embryos the basement membranes which are
most prominently stained by Alcian blue are those underlying epithelia which
are actively involved in morphogenesis - i.e. the folding neural ectoderm and
the endoderm in the region of the lateral body fold, or, where the foregut is
already closed, the ventral wall of the foregut. Others have demonstrated the
presence of GAG, including HA, in embryonic epithelial basement membranes
from older embryos and have implicated these molecules in the morphogenetic
events occurring in the epithelia associated with these basement membranes
(Bernfield, Banerjee & Cohn, 1972; Cohn, Banerjee & Bernfield, 1977; Hay&
Meier, 1974; Manasek, 1975; O'Hare, 1973).
Our observations indicate one important role that basement membranes
may play in tissue interactions. Basement membranes act as substrata for the
maintenance of intimate association of two tissues. In embryos injected with
hyaluronidase, the association of the head mesenchyme with head ectoderm and
neural ectoderm is affected, presumably due to the structural alteration of the
basement membrane. As shown in Figs. 2 and 6, contacts between the mesenchyme and ectoderm are lost, leaving spaces adjacent to the neural ectoderm
and subjacent to the head ectoderm. While exaggerated in size, perhaps due to
the additional clumping effect, these spaces are similar in location to those which
form normally during development to provide paths for neural-crest cell
migration. Some alteration in the composition of the basement membranes
could possibly account for the normal loss of attachments between mesenchyme
and ectoderm in these regions.
14
EM B 42
206
M. FISHER AND M. SOLURSH
HA is present not only in the basement membranes of epithelia undergoing
morphogenesis, but also in the ECM associated with many embryonic mesenchymes. Our results show that HA is present in primary mesenchyme matrix
in the chick as in the rat (Morriss & Solursh, 1978). HA is particularly apparent
in the head mesenchyme where greater intercellular space is found.
To explain the function of HA in ECM of mesenchymes (many of which are
actively migrating), Toole (1972, 1976) has postulated that HA may inhibit
extensive intercellular interactions by physically separating cells. This physical
separation could allow the cells to migrate and/or temporarily inhibit cytodifferentiation. Inhibition of cytodifferentiation by HA has so far been demonstrated only for chondrocytes (Toole, Jackson & Gross, 1972; Solursh, Vaerewyck
& Reiter, 1974), and evidence concerning the role of HA in cell migration is
still only circumstantial. In contrast, there is an increasing body of direct
evidence for the role of HA in establishing and maintaining spaces during
embryonic development. We have shown here that when young embryos are
injected with hyaluronidase and incubated in ovo for a short period of time
subsequent to the injection, a marked loss of intercellular space results. Earlier,
Toole & Trelstad (1971) showed the importance of HA in forming space in the
developing corneal stroma, and Pratt et ah (1975) showed that HA is present
in the newly formed space underlying the ectoderm through which neural crest
cells will migrate. In the rat embryo, Solursh & Morriss (1977) have correlated
the production of HA with the appearance of mesenchyme and its associated
spaces in the primitive streak stage, and have shown HA to be present in the
matrix underlying the neural folds (Morriss & Solursh, 1978). Experiments are
in progress to study further the importance of HA in forming and maintaining
spaces as well as the importance of the spaces themselves in development of the
chick embryo.
This investigation was supported by NTH grant HDO5505 to M. S. and USPHS training
grant no. HD-00152 from the National Institute of Child Health and Human Development,
while M.F. was a predoctoral trainee.
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FISHER,
(Received 12 April 1977, revised 29 June 1977)
14-2