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Development 113, 339-351 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
339
Expression of syndecan, a putative low affinity fibroblast growth factor
receptor, in the early mouse embryo
A. E. SUTHERLAND1, R. D. SANDERSON2*, M. MAYES1, M. SEIBERT1, P. G. CALARCO1,
M. BERNFIELD2 t and C. H. DAMSKY1
1
2
Departments of Anatomy and Stomatology, University of California San Francisco, San Francisco, California 94143, USA
Department of Pediatrics, Stanford University School of Medicine, Stanford University, Stanford, California 94305, USA
* Current address: Department of Pathology and The Center for Musculoskeletal Research, University of Arkansas for Medical Sciences.
Little Rock, AR 72205, USA
tCurrent address: Joint Program in Neonatology, Harvard Medical School, Boston, MA 02115, USA
Summary
Syndecan is an integral membrane proteoglycan that
binds cells to several interstitial extracellular matrix
components and binds to basic fibroblast growth factor
(bFGF) thus promoting bFGF association with its highaffinity receptor. We find that syndecan expression
undergoes striking spatial and temporal changes during
the period from the early cleavage through the late
gastrula stages in the mouse embryo. Syndecan is
detected initially at the 4-cell stage. Between the 4-cell
and late morula stages, syndecan is present intracellularly and on the external surfaces of the blastomeres but
is absent from regions of cell-cell contact. At the
blastocyst stage, syndecan is first detected at cell-cell
boundaries throughout the embryo and then, at the time
of endoderm segregation, becomes restricted to the first
site of matrix accumulation within the embryo, the
interface between the primitive ectoderm and primitive
endoderm. During gastrulation, syndecan is distributed
uniformly on the basolateral cell surfaces of the
embryonic ectoderm and definitive embryonic endoderm, but is expressed with an anteroposterior asymmetry on the surface of embryonic mesoder.m cells,
suggesting that it contributes to the process of mesoderm
specification. In the extraembryonic region, syndecan is
not detectable on most cells of the central core of the
ectoplacental cone, but is strongly expressed by cells
undergoing trophoblast giant cell differentiation and
remains prominent on differentiated giant cells, suggesting a role in placental development. Immunoprecipitation studies indicate that the size of the syndecan core
protein, although larger than that found in adult tissues
(75 versus 69xlO 3 M r ), does not change during periimplantation development. The size distribution of the
intact proteoglycan does change, however, indicating
developmental alterations in its glycosaminoglycan
composition. These results indicate potential roles for
syndecan in epithelial organization of the embryonic
ectoderm, in differential axial patterning of the embryonic mesoderm and in trophoblast giant cell function.
Introduction
syndecan binds in vitro to extracellular matrix components such as fibrillar collagens, fibronectin and
thrombospondin, which are usually associated with
interstitial matrices (Koda et al. 1985; Saunders and
Bernfield, 1988; Sun et al. 1989). This has led to the
proposal that syndecan serves as an anchor to bind
epithelial sheets to their subjacent stroma (Bernfield
and Sanderson, 1990). Experimental evidence shows
that syndecan does play an important role in maintenance of epithelial morphology. Decreasing the level of
syndecan expression in cultured epithelial monolayers
causes them to become fusiform and fibroblastic
(Saunders et al. 19896; Kato and Bernfield, 1990), while
Syndecan is a heparan sulfate- and chondroitin sulfatebearing integral membrane proteoglycan originally
isolated from mouse mammary epithelial cells. Syndecan's binding properties suggest that it can serve as a
receptor for extracellular matrix components and
heparin-binding growth factors, while localization
studies and transfection experiments indicate that it
plays important roles both in the maintenance of adult
tissue architecture and in organogenesis (see Bernfield
and Sanderson, 1990, for review). Despite its predominance on epithelia in adult tissues (Hayashi et al. 1987),
Abbreviations: DGD, diethylene glycol distearate; bFGF,
basic fibroblast growth factor; GAG, glycosaminoglycan;
mAb, monoclonal antibody; PEG, polyethylene glycol.
Key words: extracellular matrix, fibroblast growth factor,
mesoderm, mouse embryo, syndecan.
340
A. E. Sutherland and others
increasing the level of syndecan expression in cultures
of transformed, epithelial cells that are fibroblastic
causes them to reacquire some epithelial characteristics
(Jalkanen et al. 1990; Leppa et al. 1991). In addition,
syndecan expression undergoes striking changes during
epithelial-mesenchymal transitions that are a part of
normal fetal development: it is turned on in mesenchymal cells that will become epithelial, and turned off in
epithelial cells that become mesenchymal (Thesleff et
al. 1988; Vainio et al. 1989; Fitchett et al. 1990;
Trautman et al. 1991).
Recent studies have shown that syndecan also
participates in cellular interactions with basic fibroblast
growth factor (bFGF). Syndecan binds to bFGF in vitro
(Bernfield and Sanderson, 1990; Krufka etal. 1990) and
is homologous to a low-affinity FGF receptor identified
by ligand-affinity cloning (Keifer et al. 1990). The
binding of FGF to cell surface heparan sulfate
proteoglycans is critical to its biological activity.
Treating cells with heparitinase to remove cell surface
heparan sulfate GAG, or with chlorate, to inhibit their
sulfation, prevents binding of bFGF to a high-affinity
receptor (A.C. Rapraeger, personal communication).
In fibroblasts, this prevents FGF-stimulated growth,
and in MM14 muscle cells, causes initiation of
differentiation (A.C. Rapraeger, personal communication). Expression of a high-affinity FGF receptor in
various mutant CHO cell lines results in significant
high-affinity binding only in cells that also express cell
surface heparan sulfate (Yayon et al. 1991). The role of
heparan sulfate proteoglycans was found to be more
than stabilization of bFGF, and may involve changing
the conformation of FGF in a way that allows it to
interact with high-affinity receptors (Yayon et al. 1991).
These results indicate that the ability of cells to respond
to FGF can be regulated by changes in expression of cell
surface proteoglycans (Klagsbrun, 1990).
The apparent diversity of syndecan's functional
properties may reflect alterations in its composition.
Syndecan contains both heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) chains
(Rapraeger et al. 1985) whose size and number vary
between cell types (Sanderson and Bernfield, 1988;
Kato and Bernfield, 1989). Changes in the number of
associated heparan sulfate GAG chains or in their
sulfation could alter the affinity of syndecan for bFGF,
which binds specifically to highly sulfated heparan
sulfate (Yayon et al. 1991). The ratio of heparan sulfate
to chondroitin sulfate chains bound to the syndecan
core protein is modulated by cells in response to the
growth factor TGF-/3 (Rasmussen and Rapraeger, 1988)
suggesting one mechanism by which syndecan's activity
or specificity could be modified. Thus, syndecan has the
potential to promote signal transduction by one class of
growth factors (FGFs), and to be structurally, and
perhaps functionally, regulated by another class of
growth factors (TGF-jSs).
Some of the earliest events in mammalian embryogenesis include the organization of epithelia, such as the
trophectoderm and primitive endoderm (Fleming and
Johnson, 1988), and early inductive interactions that
may involve members of the FGF family of growth
factors (Slack et al. 1990). To help determine what role
syndecan might play in early development, we examined its distribution in mouse embryos between the 2cell and late gastrula stages, using monoclonal and
polyclonal antibodies that recognize the core protein of
the proteoglycan (Jalkanen et al. 1985; Hayashi et al.
1987; Jalkanen et al. 1988). We find that syndecan is
expressed from the 4-cell stage on, and has distribution
patterns in the postimplantation embryo that suggest
roles in stabilization of epithelia, in placental development, and in early axial patterning of the mesoderm.
Materials and methods
Materials
Protein A-Sepharose and CNBr-activated Sepharose 4B
were obtained from Pharmacia, Piscataway, NJ. Trypsin was
obtained from Difco, Detroit, MI. Diaminobenzidine, pancreatin, polyethylene glycol 1450 and 3350, the protease
inhibitors
benzamidine,
phenylmethylsulfonylfluoride,
A'-ethylmaleimide and pepstatin, and Tween 20 were from
Sigma Chemicals, St Louis, MO. Dulbecco's modified Eagle's
medium (DME) was from the UCSF Cell Culture Facility.
Diethylene glycol distearate and conical micromolds were
from Polysciences, Warrington, PA. Normal donkey serum,
biotinylated secondary antibodies to rabbit IgG, and
streptavidin-Texas Red were from Amersham, Arlington
Heights, 1L. Biotinylated secondary antibodies to rat and to
rabbit IgG and horseradish peroxidase detection kit were
obtained from Vector Laboratories, Burlingame, CA. Protein
relative molecular mass standards were obtained from
Bethesda Research Laboratories, Gaithersburg, MD, and
included
myosin
(205xl0 3 M r ),
phosphorylase
b
3
(97.4xlO Mr), bovine serum albumin (68xl0 3 M r ), ovalbumin (43xlO3Mr), and carbonic anhydrase (29xlO3Afr).
Gene-Trans was from Plasco, Inc., Woburn, MA. Heparitinase and chondroitin sulfate ABC lyase were from Miles
Laboratories, Naperville, IL.
Antibodies
The rat monoclonal antibody (mAb) to syndecan, 281-2, was
purified from hybridoma culture supernatant (Jalkanen et al.
1985), and the rabbit polyclonal anti-syndecan IgG (Jalkanen
et al. 1988) was purified from the antiserum using protein
A-Sepharose. Control antibodies included the rat monoclonal antibody MEL-14, which recognizes the murine
peripheral lymph node lymphocyte homing receptor (Gallatin
et al. 1983; and is isotype matched to 281-2 (a gift of Dr Irving
Weissman, Stanford University, Stanford, CA), and nonimmune rabbit IgG purified from serum using protein
A-Sepharose.
Embryo culture
Embryos were flushed at the 2-cell stage from superovulated
female ICR mice (12 weeks old; Harlan Sprague-Dawley Inc.)
and cultured as described previously (Sutherland et al. 1988).
Unattached hatched blastocysts were obtained by culturing
embryos at the unhatched, expanded blastocyst stage in
serum-free Eagle's medium (Spindle and Pedersen, 1973;
Spindle, 1980) in organ culture dishes for 48 h. Ectoplacental
cone (EPC) explants were made by removing 7.5-day
embryos from their implantation sites, dissecting away
Reichert's membrane, separating the embryo and ectoplacen-
Syndecan in early mouse embryos
tal cone, and then incubating the latter tissue in 0.5%
trypsin-2.5% pancreatin for 15min at 4°C to isolate the
diploid core from the surrounding giant cells. After pipetting
through a fine-bore pipette, the isolated cores were cultured
for 2-5 days in DME H-21 medium containing 10% fetal
bovine serum, during which time they differentiate and form
monolayers of giant cells.
Immunodetection in preimplantation embryos
Preimplantation embryos were fixed in Carnoy's fixative
(6:3:1 of ethanol, chloroform and glacial acetic acid) for
30min, and dehydrated and embedded in polyethylene glycol
(PEG) according to the methods of Watson and Kidder
(1988). The PEG-embedded embryos were sectioned at l^m
and mounted on slides coated with 0.1% poly-L-lysine. For
staining, the sections were rehydrated, rinsed with 0.1%
Tween 20 in phosphate-buffered saline (PBS-TW), and then
incubated in 1 % normal donkey serum in PBS-TW. Primary
antibody incubation was for 1 h at room temperature and was
followed by an overnight rinse in PBS-TW at 4°C. The
sections were then incubated in biotinylated secondary
antibody followed by streptavidin-Texas Red. The slides
were examined and photographed using Kodak Tri-X film.
Immuno detection in postimplantation embryos
Pregnant female mice, obtained as described above, were
housed until the appropriate day of gestation. For 5.5-day
embryos, the uterus was cut into sections, each of which
contained an implantation site, and fixed without further
dissection. For 6.5-day, 7.5-day and 8.5-day embryos,
implantation sites were removed from the uterus, and the
embryos were dissected out. All embryos were fixed for
45min in Carnoy's fixative, dehydrated and embedded either
in paraffin or in diethylene glycol distearate (DGD; Capco
and McGaughey, 1986; Valdimarsson and Huebner, 1989).
DGD sections (1 ,wm) were mounted on poly-L-lysine-coated
slides, rehydrated, processed and examined for immunofluorescence as described above for polyethylene glycol sections.
Paraffin sections were cut at 5 /im, mounted on slides coated
with chrome alum-gelatin, deparaffinized in xylene and
rinsed in 100% ethanol. Endogenous peroxidase activity was
quenched by incubation in 1 % hydrogen peroxide in
methanol for 30min at room temperature. The paraffin
sections were incubated in 1 % normal donkey serum in PBS
followed by an overnight incubation in primary antibody at
4°C. The sections were then incubated with biotinylated
secondary antibody, followed by streptavidin-horseradish
peroxidase. Finally they were reacted with diaminobenzidine
to develop color, the intensity of which was enhanced by
incubation in a 0.5% solution of CuSO4 in 0.9% NaCl. The
resulting slides were photographed using Kodak Technical
Pan film and a blue filter.
Radiolabelling of embryos
Hatched blastocysts were cultured overnight in serum-free,
low-sulfate Eagle's medium (with MgCl2 substituted for
MgSO4) containing 200,uCimr1 of [35S]H2SO4 (ICN Radiochemicals; specific activity 43Cimg~1 S). The labeled
embryos were then collected in a microfuge tube, frozen in
liquid nitrogen and stored at —80°C until use.
For labeling, ectoplacental cone explants were cultured
overnight on glass coverslips (12 mm diameter) in low-sulfate
Eagle's medium containing 10% fetal calf serum and
200jiCimr 1 of [35S]H2SO4, which were then frozen in liquid
nitrogen and stored at —80°C until use.
341
Proteoglycan isolation
Syndecan was isolated from labeled or unlabeled blastocysts
and outgrowths as described previously for other tissues
(Sanderson and Bernfield, 1988). Briefly, syndecan was
extracted from embryos in lml extraction buffer (10 mM Tris,
pH7.4, containing 150ITIM NaCl, 1% Triton X-100, 0.5M
KC1,1 mM phenylmethylsulfonylfluoride, 5 mM A?-ethyl maleimide, 5mM benzamidine-HCl, and lOjUgmP1 pepstatin) and
isolated using mAb 281-2 conjugated to Sepharose 4B beads.
The beads with bound proteoglycan were pelleted, rinsed and
resuspended either in digestion buffer (for enzymatic digestion of the GAG chains on unlabeled samples) or in Laemmli
sample buffer (for SDS-PAGE of 35S-labeled samples). GAG
chains were removed from the core protein by incubating the
bead-bound syndecan proteoglycan in a mixture of heparitinase and chondroitinase-ABC for l h at 37 °C as described
previously (Sanderson and Bernfield, 1988). Whole proteoglycan or isolated core protein samples were run on either
3.8—15 % or 3.5-20% polyacrylamide gradient gels. For
whole proteoglycan, the gels were fixed, dried and exposed to
X-ray film. Core protein samples were electrophoretically
transferred to Gene-Trans, a cationic nylon filter, which was
incubated with 125I-labeled mAb 281-2 and then exposed to
film.
Results
Immuno detection of syndecan in preimplantation
embryos
Syndecan was first detected, using polyclonal antisyndecan antibodies, in 4-cell embryos. From the 4-cell
stage to the 16-cell morula, it was found intracellularly
and on the external surface of the blastomeres, but not
in areas of cell-cell contact (Fig. 1A-C). By the late
morula stage (16-32 cells), syndecan was detected in a
few areas of intercellular contact, often at the interface
between outer and inner cells (Fig. ID). In the
unhatched blastocyst (120 h post-hCG), staining for
syndecan was seen in regions of intercellular contact
throughout the embryo, as well as on the external
surfaces of the trophectoderm (Fig. IE).
A major reorganization in syndecan distribution
occurred with the segregation of primitive endoderm
from the inner cell mass at the late blastocyst stage.
Syndecan became restricted to the interface of the
primitive ectoderm and primitive endoderm with some
discontinuous patches on the blastocoele surface of the
trophoblast cells, and was no longer detectable on the
external surface of the trophoblast (Fig. 1G). Intracellular staining for syndecan was detected in the
primitive ectoderm (Fig. 1H,I). The monoclonal antibody 281-2 did not detect syndecan in preimplantation
embryos prepared for immunohistochemistry under the
conditions used, suggesting that its epitope is inaccessible at these early stages.
Differential expression of syndecan along the
anterior-posterior axis in postimplantation embryos
In the embryonic region syndecan staining of
ectoderm and definitive endoderm is uniform
throughout the embryo
In the embryonic ectoderm of the postimplantation
342
A. E. Sutherland and others
Fig. 1. Sections of PEG-embedded mouse embryos showing distribution of syndecan during the preimplantation stages.
Sections of late 2-cell (A), 4-cell (B), 8-cell (C), morula (D), unhatched blastocyst (E), and hatched blastocysts (G), all
stained with polyclonal anti-syndecan antibodies. The staining is both intracellular and on the blastomere surface during the
4- to 16-cell stages (B-D), and then becomes detectable at cell boundaries at the late morula and blastocyst stages (D, E).
(icm, inner cell mass; te, trophectoderm). (F) Phase micrograph of E. After endoderm segregation (G,I), syndecan is
localized to the interface between the primitive ectoderm (pec) and primitive endoderm (pen), with patches of staining also
seen on the blastocoelic surfaces of the trophoblast cells (arrowheads, H). Intracellular staining is seen in the primitive
ectoderm (I). (H) Phase micrograph of the same embryo shown in G. The boundary between the primitive ectoderm and
primitive endoderm is marked with arrowheads. (J) Late blastocyst stained with control normal rabbit IgG. Scale
bar=25,um.
embryo (5.5-8.5 days), syndecan was expressed on the
basolateral surfaces of all cells at all stages examined;
no regional differences in staining were detected
(Figs 2, 3, and 4). Syndecan was not detectable in the
visceral endoderm (Figs 2, 3, and 4), but was expressed
by the squamous definitive embryonic endoderm that
replaces the visceral endoderm in the embryonic region
during gastrulation (Fig. 4). As in the embryonic
ectoderm, syndecan was distributed on the basal and
lateral cell surfaces of the definitive embryonic endoderm and no regional differences in its expression were
detected.
Syndecan staining displays an anterior/posterior
asymmetry in the embryonic mesoderm
Posterior and lateral mesoderm stained more intensely
Syndecan in early mouse embryos
R
343
Fig. 2. Sections of a paraffinembedded 5.5 day embryo
stained with polyclonal
antibodies to syndecan (A),
and with control normal rabbit
IgG (B). Syndecan
immunoreactivity can be seen
at the interface of the visceral
endoderm (ven) and
embryonic ectoderm (arrows),
between the cells of the
embryonic ectoderm (ec), and
in the trophoblast giant cells
(gc). No staining is seen in the
visceral (ven) endoderm or in
the extraembryonic ectoderm
(xec). Scale bars=50,um.
for syndecan than did anterior and mid-dorsal mesoderm. Syndecan was expressed by cells in the primitive
streak, and in lateral and distal mesoderm in the 7.5-day
embryo (Fig. 4A and B). In contrast, syndecan staining
was barely detectable in the anterior mesoderm of the
head fold (Fig. 4C) and in the head process and its
derivative, the notochordal plate (Fig. 5). The head
process originates at the anterior tip of the primitive
streak and gives rise to the notochordal plate during
primitive streak regression (Tarn and Meier, 1982;
Poelmann, 1981). The same asymmetry of syndecan
expression was seen in the 8.5-day embryo; syndecan
was strongly expressed in the primitive streak, in
presomitic mesoderm and in fully formed somites, but
was undetectable in the mesenchyme underlying the
anterior neural ectoderm (Fig. 6). The change in
expression was fairly abrupt, and occurred at the level
of the otic sulcus (Fig. 6A). The asymmetry in syndecan
staining in the mesoderm was confined to the embryonic region; staining for syndecan was uniformly
intense in all areas of the extraembryonic mesoderm
(Fig. 8A).
Fig. 3. Embryonic region of a section of DGD-embedded
6.5-day embryo stained with polyclonal antibodies to
syndecan. The ectoderm (ec) expresses syndecan while the
visceral endoderm (ven) does not. A few mesodermal cells
are visible (arrow) which do stain for syndecan, but less
strongly than the ectoderm. Scale bar=10jum.
In the extraembryonic region, syndecan staining is
associated with trophoblast giant cell differentiation
Both primary and secondary trophoblast giant cells
stained intensely for syndecan at all stages examined
(Figs 7 and 8). The secondary giant cells form in the
ectoplacental cone, where they differentiate from a
central core of replicating diploid cells (reviewed in
Rossant, 1986). Most, but not all, cells in the center of
the core showed no staining for syndecan, while an
annulus of cells in the zone between the central core
cells and the more peripheral differentiated giant cells
showed the most intense staining (Figs 7 and 8). The
extent of staining of central core cells appeared to be
age-related. In general, there was little or no staining in
344
A. E. Sutherland and others
the central core cells of younger embryos (6.5 days) and
increased staining in central core cells of older embryos
(7.5-8.5 days), perhaps reflecting the gradual terminal
differentiation of the cells in the core.
Syndecan was not detected in extraembryonic ectoderm cells at 6.5 days (Fig. 7). By 7.5 days, it was
detectable in those extraembryonic ectoderm cells that
contributed to the chorion as well as at the interface of
Syndecan in early mouse embryos
Fig. 4. Parasagittal sections of a DGD-embedded 7.5-day
embryo showing polyclonal antibody staining for syndecan
in the embryonic region. (A) Lower magnification view
showing staining for syndecan in the ectoderm (ec),
mesoderm (mes), and definitive embryonic endoderm (en)
as well as in the amnion (am). The boxes marked b and c
indicate the regions shown at higher magnification in
panels B and C, respectively, which were taken from
adjacent sections on the same slide. A, anterior end; P,
posterior end. (B) Posterior region, showing the three germ
layers and a small part of the amnion and extraembryonic
mesoderm. The mesodermal cell surfaces are uniformly
stained while the ectoderm is stained on basal and lateral
cell surfaces. The endoderm in this region is
morphologically more like the extraembryonic endoderm
just above, and does not stain significantly (ven).
(C) Anterior region, showing the incipient head fold.
Syndecan is present on the basal and lateral surfaces of
embryonic ectoderm and definitive embryonic endoderm
cells, but there is little detectable staining on mesodermal
cells. Scale bars=20,um.
the two layers of the chorion (Fig. 8C). Syndecan
staining was detected at the interface of the embryonic
ectoderm and overlying visceral endoderm at 7.5 days.
Since no staining was detected in visceral (extraembryonic) endoderm at any stage, the syndecan present in
this location is likely produced by the extraembryonic
ectoderm and localized at the interface of the two cell
layers.
Biochemical characterization of embryonic syndecan
The striking temporal changes in the pattern and
intensity of syndecan staining that occurred during periimplantation development suggested that temporal and
spatial modulation of syndecan structure might be
occurring. We examined this possibility by determining
the relative molecular mass of both the syndecan core
protein and the intact proteoglycan in hatched blastocysts and in isolated ectoplacental cone.
The core protein immunoisolated from hatched
blastocysts migrated as two bands of about 75xlO3Mr
and 46xlO3Mr (Fig. 9A, lane 1) while the core protein
from 72 h embryo outgrowths ran as a single band at
75xlO3Mr (Fig. 9A, lane 2). Syndecan core protein
isolated from a variety of adult epithelial tissues
migrates as a single band of 69 x 103 Mr (Sanderson and
Bernfield, 1988). Since syndecan is known to be
modified by oligosaccharides as well as by glycosaminoglycans. the difference in the apparent molecular mass
between the embryonic and adult forms may be due to
variation in the composition of these oligosaccharides
(Weitzhandler et al. 1988). The 46xlO3Mr band
detected in hatched blastocysts likely represents the
core protein of the cleaved ectodomain of syndecan,
which lacks the cytoplasmic and transmembrane
domains (Jalkanen et al. 1987).
The intact proteoglycan from hatched blastocysts ran
as a continuous region of radioactivity extending from
about 70xl0 3 M r to the top of the gel (>300xl0 3 M r ),
with some additional material not entering the gel (lane
2). In contrast, syndecan precipitated from isolated,
345
cultured ectoplacental cone ran as a more discrete
region of radioactivity extending from 200-260 xlO 3 M r
(lane 4). These results indicate that syndecan structure
is modulated during peri-implantation development.
Discussion
Syndecan has multiple distinct activities in vitro: it acts
as a receptor for several interstitial matrix molecules
(Koda et al. 1985; Saunders and Bernfield, 1988; Sun et
al. 1989), it plays a role in organization of cells into
epithelial sheets (Saunders et al. 1989a; Jalkanen et al.
1990), it is nearly identical to a hamster low-affinity
FGF receptor recently identified by ligand-affinity
cloning (Keifer et al. 1990), and binds bFGF (Bernfield
and Sanderson, 1990; Krufka et al. 1990). Determining
which of these properties are important for particular
developmental events is speculative, but can be
approached through a comparison of syndecan expression patterns with known syndecan functions.
Syndecan distribution in the late blastocyst and in
embryonic ectoderm suggests a role in epithelial
organization
The distribution of syndecan in the late blastocyst and
in the embryonic ectoderm of the early postimplantation embryo is consistent with a role in cell—cell or
cell-matrix interactions that may stabilize epithelial
morphology. Starting with endoderm segregation at the
late blastocyst stage, syndecan becomes localized to the
interface between embryonic ectoderm and visceral
endoderm. This pattern is identical to that of the first
basement membrane of the embryo, as shown by the
distributions of fibronectin, laminin and collagen type
IV (Zetter and Martin, 1978; Wartiovaara et al. 1979;
Leivo et al. 1980; Wu et al. 1983). The primitive
endoderm cells are the source of the matrix, while our
data showing the presence of syndecan staining on the
cells of the embryonic ectoderm, but not the visceral
endoderm, suggest that the ectoderm is the source of
syndecan (Figs 1-4). How syndecan redistributes at the
time that the primitive endoderm segregates is not
known, but may involve selective shedding of the
syndecan ectodomain from the apical plasma membrane and retention of intact syndecan at the basal
surface. Selective shedding of syndecan has been shown
to account for its absence on the apical surfaces and its
accumulation at the basolateral surfaces of confluent
epithelial monolayers (Rapraeger et al. 1986; Jalkanen
et al. 1987). In support of this idea, the syndecan core
protein immunoprecipitated from late blastocysts (just
after endoderm segregation) contains two components:
a 75 x 103 Mr polypeptide whose size is consistent with
that of the full-size core protein, and a 46xlO 3 M r
polypeptide whose size is similar to that of the core
protein of the ectodomain alone (Jalkanen et al. 1987).
In considering to what extent syndecan promotes and
maintains epithelial organization in embryonic and
adult tissues, it is important to note that its presence is
not required for the epithelialization process in general,
346
A. E. Sutherland and others
Fig. 5. Frontal sections of DGD-embedded embryos showing details of polyclonal antibody staining for syndecan in the
head process, the primitive streak, and the notochordal plate. In the early gastrula (7.0 days; A-D), there is little
detectable syndecan in the head process (A; hp), while in a more posterior section from the same embryo, cells in the
anterior primitive streak do stain strongly for syndecan (B; ps). In the late gastrula (8.0 days; E, F), the notochordal plate
(np) also shows little staining for syndecan. Scale bars=20^m.
and its presence is not restricted to epithelial cells
during development. For example, there are no
changes in the distribution of syndecan during compaction at the 8-cell stage, nor does it become localized to
the basolateral cell surfaces of the trophectoderm, the
first epithelium formed in the embryo. Furthermore,
syndecan is not detectable in the epithelial visceral
endoderm layer, and is expressed in nonepithelial cells
such as mesoderm and trophoblast giant cells (discussed
below). Therefore, its roles must be related to specific
functions that are undertaken by the cells in which it is
expressed.
Asymmetric distribution of syndecan in the embryonic
mesoderm suggests a role in anteroposterior patterning
of mesodermal structures
Syndecan is expressed strongly on mesoderm cells even
after they have left the primitive streak, suggesting that
Syndecan in early mouse embryos
347
Fig. 6. Parasagittal section of a DGD-embedded 8.5-day embryo showing polyclonal antibody staining for syndecan in the
embryonic region. (A) In the head, there is strong staining for syndecan in the neural ectoderm (ec) but only faint staining
in the underlying mesenchyme (mes). The arrows indicate the level at which syndecan expression changes, which is
approximately the level of the otic sulcus. (B) In the posterior region, strong staining is seen in the primitive streak (ps),
the presomitic mesoderm and in the somites (s). Scale bars=50^m.
ven»- • * •
-
•?•'
ec
B
syndecan has an important function in early mesoderm.
The asymmetry of syndecan distribution in the mesoderm suggests a role in anteroposterior patterning.
Syndecan expression is weak on the most dorsal and
anterior mesodermal structures of the gastrulating
mouse embryo (head process, notoplate and anterior
head mesoderm), and strong on the more posterior and
lateral structures (primitive streak, somites). The
potential significance of these data can be appreciated
in the light of several other findings. Syndecan binds
bFGF (Bernfield and Sanderson, 1990; Krufka et al.
1990), is nearly identical to a low-affinity receptor for
FGF recently cloned from hamster (Keifer et al. 1990)
and, as a low-affinity receptor, can regulate the
biological effects of bFGF (Yayon et al. 1991; Bernfield
and Hooper, 1991; A.C. Rapraeger, personal com-
Fig. 7. Sections of paraffinembedded 6.5-day embryo
stained with mAb 281-2 against
syndecan (A), and the control
mAb MEL-14 (B). Syndecan is
expressed by the trophoblast
giant cells (gc) and the cells of
the embryonic ectoderm (ec),
but not by cells in the core of
the ectoplacental cone (epc),
the extraembryonic ectoderm
(xec) or the visceral endoderm
(ven). Scale bars=50,um.
munication). There is evidence in other developmental
systems that members of the FGF family of growth
factors play an important role in specifying the fate of
prospective mesoderm (Smith, 1989; Melton and
Whitman, 1989; Mitrani et al. 1990). For example, in
the amphibian Xenopus laevis, treatment of animal cap
explants with bFGF results in the induction of
posterior/lateral types of mesodermal structures (Slack
et al. 1987; Kimelman and Kirschner, 1987), whereas
treatment with activin or TGF-/32 results in the
induction of anterior/dorsal mesodermal structures
(Smith, 1987; Rosa et al. 1988; reviewed in Smith,
1989). The timing and mechanism of mesoderm
specification have not been established in the mouse
embryo; however, it is known that at least one member
of the FGF family, int-2, is expressed during gastru-
348
A. E. Sutherland and others
Fig. 8. Sagittal sections of a DGD-embedded 7.5-day embryo showing polyclonal antibody staining for syndecan in the
extraembryonic region. (A) Lower power view showing the strong staining in the ectoplacental cone and giant cells (gc), in
the extraembryonic mesoderm, at the interface of extraembryonic ectoderm and extraembryonic endoderm, and between
the two layers of the chorion (ch). The boxes labeled b and c mark the areas represented at higher power in panels B and
C respectively, which were taken from adjacent sections on the same slide. (B) A subpopulation of cells in the
ectoplacental cone, which form a rough annulus around the core, stain intensely for syndecan, while the extraembryonic
ectoderm is negative. (C) There is faint staining for syndecan in the extraembryonic ectoderm that contributes to the
chorion (xec), but none in the extraembryonic endoderm (xen). Scale bars=20^m.
Syndecan in early mouse embryos
349
B
200-
20097.5-
68-
97.5-
45-j
6830-
2
3
1
3
4
Fig. 9. Syndecan core protein (Panel A) and whole proteoglycan (Panel B) isolated from hatched blastocysts and cultured
EPC. Panel A. Lane 1: Core protein from 1500 hatched blastocysts. Two species are present, at 46xlO3Mr and 75xlO3Mr.
Lane 2: Core protein from 600 72-h embryo outgrowths. One species, at 75xlO3Mr, is present. Lane 3: Core protein from
mesenteric lymph nodes. The size (64x10 Mr) is slightly smaller than that seen in most mature tissues (Sanderson et al.
1989). Panel B. Immunoprecipitates obtained with mAb 281-2 (Lanes 1, 2 and 4) or the isotype matched mAb MEL-14
(Lane 3) of extracts of S-sulfate-labeled cells and embryos. Lane 1: Intact syndecan proteoglycan from normal murine
mammary gland cells. Lane 2: Intact syndecan from 3000 hatched blastocysts. Lanes 3 and 4: Intact proteoglycan from
EPC dissected from 30 7.5-day embryos and grown in culture for 5 days.
lation in the posterior primitive streak (Wilkinson et al.
1988). Taken together, these data are consistent with
the hypothesis that the asymmetric distribution of
syndecan observed in the mouse gastrula could spatially
restrict the activity of member(s) of the FGF family
involved in generating an anterior-posterior axis in
mouse mesoderm.
Syndecan distribution in trophoblast giant cells is
consistent with a role in growth factor binding
The patterns of syndecan expression in the ectoplacental cone and trophoblast giant cells are consistent with
its proposed role as a receptor for heparin-binding
growth factors (Bernfield and Sanderson, 1990; Keifer
et al. 1990; Klagsbrun, 1990). The high level of syndecan
expression by trophoblast cells may reflect a role for
such growth factors in trophoblast function during
implantation. Trophoblast giant cells are invasive,
migrating through the uterine epithelium to establish
the embryo in the stroma (Billington, 1971; Welsh and
Enders, 1987), where they then form an intricate
network of bloodspaces, and ultimately, the fetal
portion of the mature chorioallantoic placenta (Rossant
and Croy, 1985; Rossant, 1986). Trophoblast cell
function thus involves motility, protease secretion and
neovascularization, all of which are promoted by bFGF
in cultured cells (Rifkin and Moscatelli, 1989).
In summary, it is likely that syndecan has several
functions in the early development of the mouse
embryo. Its distribution in the late blastocyst and the
embryonic ectoderm of the postimplantation embryo is
consistent with a role in matrix attachment and
epithelial organization, whereas its distribution in the
embryonic mesoderm and on trophoblast giant cells is
consistent with its proposed role as a receptor for FGF
(Keifer et al. 1990; Klagsbrun, 1990; Bernfield and
Hooper, 1991). Our results, showing a difference in the
relative molecular mass of syndecan between blastocysts and trophoblast giant cells, suggest that the spatial
and temporal regulation of syndecan structure may
reflect these potentially disparate functions. Previous
studies have demonstrated that the relative molecular
mass of the intact syndecan proteoglycan differs in
simple and stratified epithelia (Sanderson and Bernfield, 1988), and changes during B-cell differentiation
(Sanderson et al. 1989) and with epithelial—
mesenchymal interactions (Boutin et al. 1988). These
size differences are due to differences in the number
and length of heparan sulfate and chondroitin sulfate
chains attached to the core protein (Sanderson and
Bernfield, 1988). Thus, variations in GAG composition
350
A. E. Sutherland and others
may determine whether the syndecan present in
different locations functions in matrix attachment,
epithelial organization and/or growth factor binding.
The data presented here on the pattern of syndecan
expression during peri-implantation development provide a basis for future experiments designed to
determine more directly its function in the early
embryo.
We thank Dr Irving Weissman for the generous gifts of
antibodies; Dr Ray Keller for use of his microscope and for
critical comments on the manuscript and Dr Gerry Kidder for
the suggestion of DGD as an embedding medium. We also
thank Dr Barry Gumbiner, Dr Mike Frohman and Dr Ann
Poznanski for critical comments on the manuscript. Mercedes
Joves provided expert technical assistance. The work was
supported by NIH grants HD22593, CA28735, HD06763 and
HD06703, and by the Arthritis Foundation.
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