/. Embryol exp. Morph. Vol. 72, pp. 39-52, 1982
Printed in Great Britain © Company of Biologists Limited 1982
Localization of receptors for
Dolichos biflorus agglutinin in early post
implantation embryos in mice.
By MOTOKO NOGUCtfl 11 , TAKEHIKO NOGUCHI2,
MAKOTO WATANABE3 AND TAKASHI MURAMATSU4
From Biological Institute, Faculty of Science, Shizuoka University,
National Institute of Genetics, Department of Pathology,
Kobe University School of Medicine, and Department of Biochemistry,
Kagoshima University School of Medicine
SUMMARY
The distribution of receptors for Dolichos biflorus agglutinin (DBA) was studied by
histochemical staining of paraffin sections with HRP- or FITC-DBA in mouse embryos at
stages ranging from 4-5 to 12-5 days post coitum. Preimplantation blastocysts did not express
DBA receptors. The receptors first appeared in primitive endoderm cells in 5-day embryos.
In 5-5- to 7-5-day embryos, both the parietal and extraembryonic visceral endoderm cells
expressed the receptors. Reichert's membrane was negative for DBA receptors. The columnar
cells of the embryonic visceral endoderm (EVE) strongly expressed the receptors, while the
flat cells at the antimesometrial pole of the EVE were negative or patchily positive. In 8- to
9-day embryos, the receptors were expressed in the epithelium of the fore- and hindgut. In
9-5- to 12-5-day embryos, the epithelial cells of various regions of the gut expressed the
receptors, although the endodermal cells of the liver and the pancreas, both derived from
the foregut, did not express them. In 8- to 12-5-day embryos both the visceral and parietal
yolk sac endoderm were positive in DBA receptors. The receptors were localized exclusively
on the free surface facing the yolk cavity or on the luminal surface and subjacent cytoplasm.
All endodermal cells which were positive are known to have absorptive activity. All other
tissues in these stages were negative in DBA receptors.
INTRODUCTION
Cell surface and extracellular material have been assigned an important role
in embryonic morphogenesis. Cell surface carbohydrates and lectin binding
sites are known to be greatly altered during early embryogenesis in the mouse
(Muramatsu et al. 1978; 1979; Stern et al 1978; Solter & Knowles, 1978;
Kapadia, Feizi & Evans, 1980; Searle & Jenkinson, 1978; Wartiovaara, Leivo
& Vaheri, 1979). Although the function of these surface molecules in morpho1
Author's address (reprints): Biological Institute, Faculty of Science, Shizuoka University,
Oya 836, Shizuoka, 422, Japan.
2
Author's address: National Institute of Genetics, Yata 1, 111, Mishima 411, Japan.
3
Author's address: Department of Pathology, Kobe University School of Medicine, 12-7chome, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
4
Author's address: Department of Biochemistry, Kagoshima University School of Medicine, 1208-1 Usuki-cho, Kagoshima 890, Japan.
5
Author to whom reprint request should be addressed.
39
40
M. NOGUCHI AND OTHERS
genesis is not well understood, lectins and antibodies recognizing certain terminal sugar sequences can be conveniently used to distinguish and trace particular
populations of cells in morphogenesis (Sobel & Nebel, 1978; Chavez & Enders,
1981).
Receptors for Dolichos biflorus agglutinin (DBA), a lectin specific for terminal
D-iV-acetylgalactosamine (Etzler, 1972), were found to be expressed on stem
cells and primitive endodermal cells of teratocarcinoma but not in variety of
other differentiated cells derived from them in vitro (Muramatsu, Muramatsu &
Ozawa, 1981).
Therefore, we are interested in the behaviour of the DBA receptor sites in
normal postimplantation development when the three germ layers are formed
and organogenesis begins.
In this report, the expression of DBA receptors in mouse embryos from 4-5
to 12-5 days post coitum (p.c.) was investigated by a fluorescent isothiocyanate
or peroxidase-labelled DBA staining technique. DBA receptors were exclusively
found in those endodermal cells showing absorptive activity.
MATERIALS AND METHODS
Mouse embryos. Embryos from 129/Sv-SlCP and C57BL/1O strains mice
were used. Copulation was determined by the presence of a vaginal plug. The
middle of the 'artificial night' was designated as day 0 of pregnancy. The
stages of the embryos ranged from 4-5 to 12-5 days p.c. Five to eight embryos
of each strain at each stage were used for each DBA staining and control.
Lectins. DBA labelled with fluorescein isothiocyanate (FITC-DBA, OD 495/
280 ratio = 1-05) or with horseradish peroxidase(HRP-DBA) were the products of E.Y. Lab., San Mateo, Cal., U.S.A.
Haptenic Sugar. D-7V-acetylgalactosamine (GalNAc) was the product of
Nakarai Chemical Co., Japan.
Preparation of specimen. Whole uterus of 4-5-5 days p.c., uterine swelling
of 5-5-8-5 days and the whole embryos of 9-5-12-5 days were recovered and
immediately fixed in cold 95% ethanol for 24 h. They were dehydrated with
ethanol, soaked in xylol, embedded in paraffin and serially sectioned as
6 ju,m.
Staining. All the sections of a single embryo were subjected to experimental
staining or to control staining when the stage was from 4-5 to 8-5 days p.c. A
part of the sections from a single embryo was used for experimental staining
and another part for control staining when the stage was from 9-5 to 12-5 days
p.c.
(i) Staining with HRP-DBA. Specimens were immersed in m e t h a n o l - 0 3 %
H2O2 for 30 min to block the activity of intrinsic peroxidase and washed with
Dulbecco's phosphate-buffered saline (DPBS). Subsequently, they were incubated in a dilute solution of HRP-lectin (100/tg/ml in DPBS) for 30 min at
DBA receptors in postimplantation mouse embryos
41
Table 1. Localization of DBA receptors in the three germ layers and their
derivatives in postimplantation mouse embryos ranging from 4-5 to 12-5 days p.c.
(The intensity of HRP-DBA stain is shown as symbol ( - ) and (+):
( - ) , negative, ( + , + +, + + +)*, positive.)
Blastocyst (preimplantation)
Trophoblast
Embryonic and extraembryonic ectoderm
Neural plate, neural tube, brain tissue
Primitive streak
Epidermis
Mesoderm
Notochord, somite, mesenchyme
Heart, kidney, cartilage, muscle, genital ridge
Primitive endoderm
Extraembryonic visceral endoderm
Embryonic visceral endoderm
Foregut epithelium
Hindgut epithelium
Intestinal epithelium
Liver, pancreas, lung
Visceral yolk sac endoderm
Parietal yolk sac endoderm
Blood island
Amnion, chorion, allantois
—
—
—
—
—
+
+++
+ ->• — and +
— -> + +
—->•+ +
++
—
+++
++
—
* Examples of the notation ( + , + + , + + +) are seen in the Figures listed below.
(+) VE and PE endoderm cells indicated by arrows in Fig. 2 and flat cells in EVE endoderm cells in Fig. 3.
(+ +) HG endoderm cells indicated by arrows in Fig. 7 and the intestinal epithelial cells
in Fig. 10.
(+ + +) VYS endoderm cells in Fig. 7.
room temperature in a moist chamber. After incubation, each section was
washed thoroughly in DPBS and stained with 3,3'-diamino azobenzidine (DAB)
in 0-05 M-tris-HCl, pH 7-6. Some sections were stained with haematoxylin.
(ii) Staining with FITC-DBA. Deparaffinized sections were incubated with
FITC-DBA (100 /Ag/ml in DPBS) for 30 min in a moist chamber. Subsequently,
they were washed in DPBS thoroughly and mounted in fresh DPBS. The
specimen was observed using an Olympus fluorescence microscope equipped
with a mercury lamp HBO 200W and Olympus filter system BV and Y-50.
(iii) Control Staining. Specimens were incubated with HRP-DBA or FITCDBA diluted in DPBS containing 0-2 M-GalNAc. All other procedures in control
staining were the same as those in experimental staining.
42
M. NOGUCHI AND OTHERS
,-TE
1
Fig. 1. Absence of DBA receptors in the 4-5-day embryo in the uterus. HRPDBA stain. 40 x 3-3. This blastocyst is peri-implantation stage, and some primitive
endoderm cells have been formed already (-»). DBA receptors are not expressed in
both the embryoblast (E) and the trophectoderm (TE).
RESULTS
The distribution of DBA binding sites was investigated by staining with
HRP- or FITC-DBA in mouse embryos at developmental stages ranging from
4-5 to 12-5 days p.c. The results of HRP-DBA stain were apparently identical
to those of FITC-DBA on each stage. Therefore, the results will be documented
by using the sections stained with HRP-DBA, since the structural presentation
is better in HRP-DBA stained sections than in FITC-DBA stained ones.
As summarized in Table 1, DBA receptors were detected only on the cells of
endodermal lineage in both 129/Sv-SlCP and C57BL/10 strains mice. DBA
receptors were present in the parietal endoderm at every stage examined.
Reichert's membrane was DBA receptor negative. In the visceral endoderm,
the extent of expression of DBA receptors varied with stage. Precise observation
on each stage will be described below.
4-5- to 5-day embryos. Some blastocysts were in preimplantation stage and
others were beginning implantation in 4*5 days/?.c. Some of them have differentiated the primitive endoderm cells. All 4-5-days blastocysts were negative for
DBA receptors (Fig. 1). DBA receptors were first recognized in the primitive
endoderm cells only weakly in 5-day embryos. Both trophoblast and the
embryonic ectoderm were negative for the receptors.
DBA receptors in postimplantation mouse embryos
43
PE
VE
EVE
ii
Fig. 2. Localization of DBA receptors in the 5-5-day embryo in the uterus. HRPDBA stain with haematoxylin poststaining. 40x5. DBA receptors have a patchy
distribution (-•) in both the visceral endoderm (VE) and the parietal endoderm
(PE). Some cells stain only weakly (-•). Receptors can be seen both on the cell surface
and in the cytoplasm. Both the extraembryonic (EXEC) and the embryonic (EEC)
ectoderm are negative for DBA receptors.
Fig. 3. Localization of DBA receptors in the 6-5-day embryo in the decidua. HRPDBA stain. 10 x 3-3. Both the extraembryonic visceral (EXVE) and the parietal (PE)
endoderm cells are positive for DBA receptors (->). In the embryonic visceral
endoderm (EVE), the columnar cells stain strongly (•+), but the flat cells stain only
weakly. The EXEC and EEC ectoderm cells, Reichert's membrane (RM) and
trophoblast (TR) are negative for DBA receptors.
5-5- to 6-5-day embryos. After the formation of the visceral and parietal
endoderm in 5-5-day embryos, DBA receptors had a patchy distribution in
both types of endodermal layers. The lectin receptors appeared to be located
in the cytoplasm (Fig. 2). In 6-5-day embryos the extraembryonic visceral endoderm cells were tall and columnar (Fig. 3). In these cells, the receptors were
strongly expressed on the free surface facing the yolk cavity, but not on the
surface facing the extraembryonic ectoderm. At this stage, the embryonic
visceral endoderm cells become flat, and DBA receptors were rather weakly
expressed on the free surface of these cells. On the other hand, the columnar
ones near the extraembryonic visceral endoderm stained ^ongly. All other
cells of the egg cylinder and trophoblasts were negative tor DBA receptors.
7- to 7-5-day embryos. In 7- to 7-5-day embryos in which mesoderm formation
has begun, the extraembryonic visceral endoderm cells stained strongly (Fig.
4, 5). In these cases, the free cell surface stained. On the other hand, the embryo-
44
M. N O G U C H I AND OTHERS
EXVE
TR
Fig. 4. Localization of DBA receptors in the 7-day embryo in the decidua. HRPDBA stain. 10 x 5. Localization of DBA receptors is almost the same as that in
the 6-5-day embryo in Fig. 3. (-»). Note the heavy stain on the free surface of the
columnar cells of EVE (-•). Mesoderm (M) is negative.
Fig. 5. Localization of DBA receptors in the 7-5-day embryo in the decidua. HRPDBA stain. 10 x 5. The columnar cells in the EXVE and EVE and some of the
flat cells in EVE stain strongly (-»). Note distinct stain on the free surface of these
cells facing yolk cavity (YC). The neural plate (N), mesoderm (M) and the extraembryonic membranes such as the amnion (A) or the chorion (C) are negative.
nic visceral endoderm cells in 7-day embryos still remained as flat as those of
6-5-day embryos, and either stained weakly or did not stain at all. In 7-5-day
embryos, some cells of the embryonic visceral endoderm stained as strongly as
the extraembryonic visceral endoderm cells, but such cells were rare. The
parietal endoderm cells did not stain so strongly as the extraembryonic visceral
DBA receptors in postimplantation mouse embryos
45
endoderm cells. The mesodermal cells, the amnion and the chorion did not
stain with DBA.
8- to 8-5-day embryos. At these age, the foregut pocket and the hindgut
pocket have begun to form and become deeper. The luminal surface of the
columnar cells of the foregut and the hindgut expressed the receptors more
intensively than those of the endodermal cells on the outside of the embryos
(Fig. 6, 7). The visceral yolk sac (VYS) and parietal yolk sac (PYS) endoderm
cells also remained strongly positive for DBA receptors. The stained part was
confined to the surface lining the yolk cavity. At larger magnification one could
see that DBA receptors were distributed around both the rough surface of
VYS cells and rather smooth surface of PYS cells. A large number of heavily
stained vesicles were also observed in the apical cytoplasm of VYS endoderm
cells (Fig. 8). Blood islands, the allantois, and other mesodermal and ectodermal cells were all negative.
9- to 12-5-day embryos. In these stages, new interactions occur in the three
germ layers and organ rudiments emerge and organogenesis is in progress.
Even in these stages, positive cells were confined to tissues of endodermal
lineage. In 9-day embryos, the fore- and hindgut form a continuous tube,
although the midgut remains open to the yolk cavity in the middle of the embryo.
DBA receptors were strongly labelled in the columnar epithelium extruding
from the portal region to the floor of the fore- and hindgut. On the other hand,
DBA receptors were still negative in the flat cells of the dorsal epithelium of
these fore- and hindgut (Fig. 9).
In 9-5- to 12-5-day embryos the fore- and hindgut undergo transformation
into various kinds of digestive organs and the derivative organs such as lung
and thyroid. In these stages, DBA receptors were expressed in the columnar
epithelium of the various gut regions (Fig. 10). The luminal free surface was
positive, but not so strongly as that of adult intestine. All other endodermal
epithelial cells such as those of the primitive liver, pancreas and lung were
negative for DBA receptors. Other tissues positive for DBA receptors were
VYS and PYS endoderm. Again, only the luminal free surface stained strongly
and the superficial cytoplasm contained DBA-positive granules.
The mesodermal tissues such as the primitive heart or kidney and the ectodermal tissues such as the primitive epidermis or nervous tissues were all
negative.
Control. The specificity of the reaction was confirmed by preincubation of
lectin with jV-acetylgalactosamine. All specimens of the embryos at the stages
ranging from 4-5 to 12-5 days/7.c. were unstained (Fig. 11).
46
M. NOGUCHI AND OTHERS
A
i»
t-VYS
%
TB:
AL
Cx
PYS
DBA receptors in postimplantation mouse embryos
47
DISCUSSION
In mouse embryos at stages ranging from 4-5 to 12-5 days p.c, the cells
positive for DBA receptors were those of endodermal lineage. Although in
very primitive endoderm cells of blastocysts in peri-implantation stage the
receptors were almost negative, the receptors were expressed, with increasing
developmental age, on the primitive endoderm cells, on the extraembryonic
visceral endoderm cells, on some of the embryonic visceral endoderm cells, on
the parietal endoderm, and on VYS and PYS endoderm, and finally on some
of the primitive gut epithelia. During early uterine life, DBA receptors were
rarely detected on the cells of either ectodermal or mesodermal lineages.
Although most of these cells of endodermal lineage possessed receptors, intensity and cellular localization of the DBA receptors varied with cell type.
Electron microscopical observations have suggested that embryonic and
extraembryonic visceral endoderm cells have intensive absorptive and nutritional functions (Solter, Damjanov & Skreb, 1970; Batten & Haar, 1979).
Numerous pinocytotic vacuoles, microvilli, primary and secondary lysosomes,
and fair amounts of rough endoplasmic reticulum and free ribosomes are the
most important characteristics of these cells. Endoderm cells of VYS contain
specialized endocytic systems and microvilli lining the yolk cavity (Lambson,
1966; Schliiter, 1978; Takeuchi, 1980). Ultrastructurally PYS endoderm cells
appear spherical or oval, with regular and smooth outlines, microvilli being
absent until the middle stage of gestation (Jollie, 1968; Jensh et ah, 1977).
The endocytic systems on the surface and in the subjacent cytoplasm of these
cells suggest that these cells phagocytose the material which passes through
Reichert's membrane from the maternal blood (Jollie, 1968; Carpenter, 1980).
Fig. 6. Localization of DBA receptors in the foregut pocket in the 8-5-day embryo.
HRP-DBA stain. 10x5. Note appearance of DBA receptors on the luminal surface
of the endoderm cells near the opening of the foregut pocket (FG) (-»•). Both the
visceral yolk sac (VYS) and parietal yolk sac (PYS) endoderm cells stain strongly.
Other primitive organs such as somites (SM), the heart (H), (A) and (TR) are
negative.
Fig. 7. Localization of DBA receptors in the hindgut pocket in the 8-5-day embryo.
HRP-DBA stain. 10 x 5. DBA receptors appear on the luminal surface of the
endoderm near the opening of the hindgut pocket (HG) (-»). The embryonic endoderm cells on the outside are still negative. Both VYS and PYS endoderm cells
stain strongly. The allantois (AL), amnion (A), the neural tube (NT) and mesoderm
(M) are negative.
Fig. 8. Localization of DBA receptors in both VYS and PYS endoderm at larger
magnification. HRP-DBA stain. 40 x 5. DBA receptors are strongly expressed
around both the rough cell surface of VYS endoderm and rather smooth surface of
PYS endoderm. Note a large number of heavily stained vesicles (O) in the apical
cytoplasm of VYS endoderm cells. Reichert's membrane (RM) and the blood
islands (BI) are negative.
48
M. NOGUCHI AND OTHERS
NT
AL
> I
E"
MC
IE .
MC
10A
I .•>/..
10B
-
MC
DBA receptors in postimplantation mouse embryos
49
Fig. 11. An example of control staining of DBA receptors. The 7-day embryo
stained by HRP-DBA with GalNAc. 20 x 3-3. All cells are unstained.
Results documented in this communication reveal that DBA receptors are
present most strongly on the luminal surface of these cells and in their apical
cytoplasm which contain the endocytic systems. The intensity of the expression
in PYS endoderm was not so strong as VYS endoderm. This might be due to
the absence of well developed microvilli in PYS endoderm cells.
As the mesoderm formation advances, the embryonic visceral endoderm cells
become flattened and depleted of microvilli and of almost all endocytic systems
(Solter et al 1970).
Fig. 9. Localization of DBA receptors in the hindgut in the 9-day embryo. HRPDBA stain. 10 x 5. DBA receptors are expressed on the hindgut columnar epithelium
(->) extruding from the portal region (IP) to the floor of the hindgut. The VYS and
PYS endoderm cells are positive in DBA receptors. The flat cells of the dorsal
epithelium of the hindgut are still negative. Other tissues such as NT, AL, TR
and BI are negative.
Fig. 10 A. Localization of DBA receptors in the gut in the 11-5-day embryo. HRPDBA stain with haematoxylin poststaining. 10 x 5. DBA receptors are localized in
the intestinal epithelium (IE) (->). The mesenchyme (MC) and other tissues are
negative.
Fig. 10B. Localization of DBA receptors in the 11-5-day intestinal epithelium at
larger magnification. HRP-DBA stain. 40 x 5. DBA receptors are seen on the
luminal cell surface of intestinal epithelial cells (->). The mesenchyme is negative.
50
M. NOGUCHI AND OTHERS
This study shows that expression of DBA receptors is either patchy, weak
or absent in this region, and that only the columnar cells near the extraembryonic visceral endoderm express DBA receptors. Therefore, it is possible
that columnar cells positive for the DBA receptor migrate into the extraembryonic region rather than lose the receptor, and that negative cells in this
region are undifferentiated definitive endoderm cells recruited from embryonic
ectoderm (Snell & Stevens, 1975; Gardner, 1978; Hogan & Tilly, 1981).
As the epithelium of the fore- and hindgut derived from this definitive endoderm transforms into the gut epithelial cells, the absorptive organelles such as
microyilli and endocytic systems appear on the luminal surface and the apical
cytoplasm (Overton, 1965).
These results reveal that DBA receptors are expressed on the luminal surface
of the gut epithelial cells. However, the intensity was not so strong as that seen
in the adult intestine (Watanabe, Takeda, Urano & Muramatsu, 1982), which
has a strong absorptive function (Cardell, Badenhauser & Porter, 1967; Ito,
1965). Thus, expression of DBA receptors paralleled exactly that of microvilli
and endocytic system. In contrast to the gut epithelia, DBA receptors were not
detected on parenchymal cells of the liver, lung and pancreas, all of which are
derived from the foregut endoderm.
Thus in the early half of uterine life, DBA receptors are expressed exclusively
on cells which are of endodermal origin, and have simultaneously an absorptive
function.
This parallelism of DBA receptors and an absorptive function, however, does
not hold true in cells of non-endodermal origins. For example, sperms, oocytes
and the renal collecting tubules in adult mice (Watanabe, Muramatsu, Shirane
& Ugai, 1981) and thymocytes in the 13th day foetuses (Kasai et al., 1980) have
DBA receptors. These types of cells do not seem to have any specialized absorptive function. On the other hand the renal brush border which has an intense
absorptive function does not possess DBA receptors (Watanabe et al. 1981).
During preimplantation stages a gradual fall in the extent of DBA receptor
expression has been noticed. Fujimoto, Muramatsu, Urushihara & Yanagisawa
(1982) studied distribution of DBA receptors on freshly flushed, unfixed cleaving
eggs and early blastocysts. They found that cleaving embryos intensely express
DBA receptors, and that the extent of staining becomes weaker in early blastocysts. We got similar results with embryos fixed in situ (Noguchi, M. unpublished). In late blastocysts, the receptors become undetectable by the histochemical method used in this report.
Many experimental data have suggested that stem cells of teratocarcinomas
correspond to cells of embryonic ectoderm of early postimplantation embryos
(Stevens, 1975; Evans, Lovell-Badge, Stern & Stinnakre, 1979; Martin, 1980;
Noguchi & Kume, 1980). We have observed that stem cells of certain teratocarcinomas, such as F9 cells and core cells of teratocarcinoma OTT6050,
strongly express DBA receptors (Muramatsu et al. 1981). However, the normal
DBA receptors in postimplantation mouse embryos
51
counterparts of teratocarcinoma stem cells were DBA negative. At present
there is no explanation for the discrepancy in DBA receptor expression between
the multipotential embryonic cells and teratocarcinoma stem cells.
The receptors expressed in teratocarcinomas are high-molecular-weight glycoproteins (Muramatsu et al. 1981; Ozawa, Yonesawa, Sato & Muramatsu, 1982).
As DBA reactivity is merely a marker of the presence of a terminal sugar moiety
(D-N-acetylgalactosamine), it is quite possible that DBA receptors found on
those various type of tissues may be different glycoconjugates.
In any event, DBA receptors seem to be useful markers of absorptive cells
of endodermal origin in early postimplantation embryos. This knowledge will
be especially helpful in cell separation in early postimplantation embryos.
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{Received 20 October 1981, revised 24 May 1982)