/. Embryo/, exp. Morph. Vol. 46, pp. 135-146, 1978
Printed in Great Britain © Company of Biologists Limited 1978
135
Modulation of alphafetoprotein synthesis in the
early postimplantation mouse embryo
By M. DZIADEK 1
From the Department of Zoology, Oxford
SUMMARY
The visceral endoderm of mouse egg cylinders on the 7th and 8th days of gestation is
divided into the visceral embryonic (VE) endoderm cell population which synthesizes
alphafetoprotein (AFP), and the visceral extra-embryonic (VEX) endoderm population which
does not synthesize AFP. Embryonic (E) and extra-embryonic (EX) ectoderm and visceral
endoderm tissues were enzymically separated, reassociated in different combinations, and
cultured in vitro for 48 h. The immunoperoxidase reaction on sections of cultured tissues
showed that both VE and VEX endoderm cells synthesize high levels of AFP when cultured
in isolation or in association with E ectoderm, but do not synthesize AFP when in close
association with EX ectoderm. Both 7th and 8th day VEX endoderm cells synthesize detectable levels of AFP 12 h after isolation, and contain high levels by 24 h. It is concluded that
both VE and VEX endoderm cells have the ability to synthesize AFP, but modulation of
expression occurs through an inhibitory influence of the EX ectoderm.
INTRODUCTION
Initial differentiation of cells in the early mouse embryo appears to involve
cell-cell or cell-environment interactions. After cavitation and expansion of the
blastocyst, cells on the blastocoelic surface of the inner cell mass form a layer of
primitive endoderm (Enders, 1971;Nadijcka & Hillman, 1974), apparently in
response to an 'outside' position with respect to other cells (Gardner &
Johnson, 1975; Rossant, 1975). The primitive endoderm contributes to the
visceral endoderm layer and to the parietal endoderm of Reichert's membrane
(Snell & Stevens, 1966). The parietal and visceral derivatives differ in their
synthetic properties. Parietal endoderm cells have been reported to synthesize
a plasminogen activator (Strickland, Reich & Sherman, 1976), while visceral
endoderm cells synthesize alphafetoprotein (AFP) (Wilson & Zimmerman;
1976, Dziadek & Adamson, 1978). Studies on the localization and synthesis of
AFP during mouse embryogenesis showed that AFP synthesis is a property
specific to the visceral endoderm before liver formation (Dziadek & Adamson,
1978). The immunoperoxidase reaction on tissue sections showed that AFP first
appears in some visceral embryonic (VE) endoderm cells of the 7th day embryo.
On the 8th day AFP is confined to all YE endoderm cells, while the visceral
1
Author's address: Department of Zoology, South Parks Road, Oxford 0X1 3PS, U.K.
136
M. DZIADEK
extra-embryonic (VEX) endoderm cells do not react with the anti-AFP antiserum. The visceral endoderm of 9th day embryos is also divided into AFPpositive and AFP-negative cell subpopulations, whereas all visceral endoderm
cells of the 10th day yolk sac contain AFP.
The clear-cut division of visceral endoderm cells into the AFP-positive and
AFP-negative populations in 8th day embryos was very closely related to their
positions over the embryonic (E) ectoderm and extra-embryonic (EX) ectoderm
tissues respectively. Such a correlation suggested that AFP synthesis in the
visceral endoderm may be dependant on an interaction with the underlying
tissue. The present study is an investigation of whether tissue interactions do
control AFP synthesis in the visceral endoderm of the postimplantation mouse
embryo. Control of AFP synthesis could be at two levels: at a primary level in
which the tissue acquires a capacity for AFP synthesis (AFP synthetic programme); or at a secondary modulatory level in controlling the level of expression
of a programme which is already present. E and EX ectoderm tissues were
reassociated with either VE or VEX endoderm to determine whether the
E ectoderm exerts a positive influence over AFP synthesis, or whether EX
ectoderm exerts a negative influence, and at what level these influences operate.
MATERIALS AND METHODS
Isolation of embryonic tissues
Embryos used for all experiments were the F2 progeny of either C3H x 129J
or CBA x C57BL F\ crosses. The day of the vaginal plug was designated the
first day of pregnancy. Uteri were dissected from mice on the 7th and 8th days
of pregnancy into the prewarmed phosphate-buffered saline (PBS, solution A
of Dulbecco & Vogt, 1954). Embryos were dissected from the decidual tissue
into Whitten's medium (Whitten, 1971) gassed with 5 % CO2, 5 % O2, 90 % N 2
in a humidified incubator at 37 °C. Egg cylinders were isolated from the parietal
endoderm and ectoplacental cone using tungsten needles. Two transverse cuts
were made through the centre of the egg cylinders to produce complete
embryonic and complete extra-embryonic fragments (see Dziadek and Adamson, 1978). These were incubated in a solution of 2-5 % pancreatin and 0-5 %
trypsin (Levak-Svajger, Svajger & Skreb, 1969) for 5 min at 4 °C. The layers
were separated by gentle pipetting using a glass pipette with bore diameter
slightly smaller than the diameter of the tissue fragment. Isolated E and EX
ectoderm and VE and VEX endoderm tissues were washed and cultured in
a-medium plus serum (see below) for £-1 h before reassociation to remove
residual enzyme activity.
Labelling of visceral endoderm by the uptake of melanin granules
Visceral endoderm cells can ingest melanin granules from surrounding
medium, and localization of melanin granules in sectioned tissue can be a useful
marker for this cell type. Melanin granules were prepared from the eyes of male
Modulation of AFP synthesis
137
CBA mice. Four whole eyes were dissected into approximately 2 ml of
Whitten's medium. The lenses were discarded and the heavily pigmented iris
and retinal epithelia were shredded using fine forceps, thus releasing large
quantities of melanin granules into the medium (method of R. L. Gardner,
personal communication). This suspension was pipetted from the dish after the
debris had settled. Isolated VE and VEX endoderm layers were incubated in
the suspension of melanin granules for 3 h and then washed thoroughly in
several changes of Whitten's medium. Melanin granules were freshly prepared
for each experiment.
Tissue culture
Isolated and reassociated tissues were cultured in microdrops of a-medium,
lacking nucleosides and deoxynucleosides plus 10 % heat-inactivated foetal calf
serum (Stanners, Eliceiri & Green, 1971), on bacteriological plastic Petri dishes
(Sterilin Ltd., Richmond, Surrey), under liquid paraffin oil (Boots Pure Drug
Company, U. K.). Cultures were maintained in humidified 5 % CO2 in air, at
37 °C. These culture conditions do not inhibit AFP synthesis in visceral
endoderm cells (Dziadek & Adamson, 1978).
Immunoperoxidase reaction for AFP
The preparation of the anti-AFP antiserum and the tests for its specificity
have been described in a previous report (Dziadek & Adamson, 1978). AFP
synthesis by cultured tissues was determined by the immunoperoxidase reaction
on tissue sections, using the Sainte-Marie technique for tissue fixation (SainteMarie, 1962) with Engelhardt's modification employing a mixture of 96 %
ethanol with glacial acetic acid (99:1, v/v) for the fixative (Engelhardt, Goussev,
Shipova & Abelev, 1971). Preparation of sections and the procedure for
incubation in antisera and subsequent reaction with diaminobenzidine are as
outlined previously (Dziadek & Adamson, 1978). Control incubations were
done routinely with antiserum which had been absorbed with AFP. Very low
background levels of peroxidase activity were observed in these control sections,
indicating negligible cross-reactivity of the antisera used, and the specificity of
the anti-AFP antiserum for AFP.
RESULTS
AFP synthesis in visceral endoderm cells after reassociation with embryonic and
extra-embryonic ectoderm
Cells of isolated tissues cultured in vitro can contain AFP only if they have
synthesized or are synthesizing AFP, since AFP is not present in the culture
medium for adsorption (Dziadek & Adamson, 1978). Isolated E and EX
ectoderm tissues do not contain AFP after culture, which indicates that these
tissues do not synthesize AFP and that a new layer of visceral endoderm cells
138
M. DZIADEK
MG
2A
MG
50 pim
1A
MG
2C
MG
IB
MG
2D
Modulation of AFP synthesis
139
does not reform from the isolated ectoderm (Dziadek & Adamson, 1978).
All tissues known to synthesize AFP also accumulate AFP, and it is therefore
assumed in this study that absence of accumulation indicates that the tissue
is not synthesizing AFP.
In in vitro studies on the control of AFP synthesis, it is necessary to have
a reliable marker for visceral endoderm cells, which is independent of AFP
synthesis. Visceral endoderm cells can accumulate and retain melanin granules
in their cytoplasm, which are easily recognizable in histological sections.
Preliminary studies showed that granules are taken up by 20-40 % of both VE
and VEX endoderm cells after a 3 h incubation period. Between 5 and 15
granules were initially present close to the cell membrane of each labelled cell
(Fig. 1). Immunoperoxidase staining showed AFP to be present in VE endoderm cells, but not VEX endoderm cells, immediately after incubation in
melanin granules. After 48 h in culture melanin granules were present throughout the cytoplasm of labelled cells, and both VE and VEX endoderm cells
contained high levels of AFP (Fig. 2). Melanin granules were not easily visualized
in cells with heavy peroxidase staining, but were present in adjacent control
sections. Uptake of melanin granules by visceral endoderm cells therefore does
not inhibit their ability to synthesize AFP, and can be used as a marker for these
cells in studies on AFP synthesis.
The four tissue types (E and EX ectoderm, VE and VEX endoderm) were
isolated enzymically from 8 to 12 7th day embryos for each experiment. Visceral
endoderm and ectoderm tissues were easily distinguishable under the transmittedlight microscope; the endoderm having a dark, granular appearance, and the
ectoderm appearing smoother and more translucent. Visceral endoderm tissue
layers also had the tendency to curl up after isolation. E and EX ectoderm
tissues were left intact, while the visceral endoderm layers were broken up into
smaller fragments by further pipetting. In three experiments visceral endoderm
cells were labelled with melanin granules, and in five experiments were left
unlabelled. In two initial experiments two ectoderm fragments were aggregated
with two unlabelled visceral endoderm fragments, and in all subsequent
experiments single fragments of each tissue type were aggregated. In all cases
F I G U R E S 1 AND 2
Fig. 1. Immunoperoxidase reaction on sections of VE endoderm (A) and VEX
endoderm (B) after culture in melanin granules (MG). Only VE endoderm cells
contain AFP, and melanin granules are present at the cell membrane of some cells
in both tissues.
Fig. 2. Immunoperoxidase reaction on sections of VE and VEX endoderm tissues
which were cultured for 48 h after incubation in melanin granules. Both VE
endoderm (A) and VEX endoderm (C) contain high levels of AFP. Control sections
treated with antiserum adsorbed with AFP show melanin granules (MG) present in
both VE (B) and VEX (D) endoderm cells. Calibration for B applies also for A;
Calibration for D applies also for C.
140
M. DZIADEK
Table 1. The presence ( + ) or absence ( —) of AFP in visceral endoderm cells after
culture with embryonic or extra-embryonic ectoderm for 48 h, or in isolation for
0 or 48 h
Experiments with melanin granules (MG) are indicated separately. The number of
observations are summed from all experiments and are indicated in parentheses.
Cultured in
isolation,
Oh
VE
VE+MG
VEX
VEX+MG
+ (6)
+ (8)
-(6)
- (7)
Reassociated Reassociated
with
with
E ectoderm, EX ectoderm,
48 h
48 h
+
+
+
+
(9)
(5)
(9)
(6)
-(13)
- (U)*
-(17)
- (10)
Cultured in
isolation,
48 h
+
+
+
+
(10)
(8)
(16)
(8)
* In two aggregates (not included in total) AFP-positive VE endoderm cells were
attached as a clump to the EX ectoderm (see text).
the total mass of ectoderm was about three to four times that of the visceral
endoderm. Ectoderm and visceral endoderm tissues do not reassociate readily,
and aggregation was facilitated by frequently drawing the tissues together
using fine glass pipettes, or by initially limiting the size of the culture drop to
draw the tissues together and incubating for a few hours before adding more
medium. In the majority of cases tissues had associated after 24 h in culture,
and after 48 h the visceral endoderm was observed to have grown over the
ectoderm, but in most cases did not surround it completely. Very little tissue
degeneration was observed during the 48 h culture period, after which tissue
aggregates were fixed for the immunoperoxidase reaction. Tn three experiments
representative fragments of VE and VEX endoderm were fixed at zero time, to
determine the synthetic activity of these tissues before aggregation.
The results of these experiments are summarized in Table 1. Analysis of AFP
synthesis at the start of aggregation showed that VE endoderm cells contained
AFP, whereas VEX endoderm cells were entirely unlabelled. When reassociated
with E ectoderm, both VE and VEX endoderm cells contained high levels of
AFP after 48 h in culture in all experiments (Fig. 3). In aggregates with EX
ectoderm, no AFP-positive cells were present in the majority of cases. A few
outer cells in three aggregates were observed with very low levels of AFP. In
experiments with labelled VE and VEX endoderm cells, the presence of melanin
granules in outer cells of tissue aggregates indicated the presence of visceral
endoderm cells (Fig. 4). In two cases VE endoderm cells had remained as
a clump adhering to one side of the EX ectoderm, and many of these cells were
not in direct contact with EX ectoderm tissue. Immunoperoxidase staining
showed that the outer endoderm cells contained AFP, presumably because they
were not in contact with EX ectoderm. In contrast, those visceral endoderm
cells which were labelled with melanin granules and which were in contact with
Modulation of AFP synthesis
141
EX ectoderm, were AFP-negative (Fig. 5). In a few cases VE and VEX endoderm
cells had failed to associate with EX ectoderm and remained as free vesicles in
the culture drops. All of these visceral endoderm fragments were found to
contain high levels of AFP. Both VE and VEX endoderm tissues showed very
high levels of AFP after culture in isolation for 48 h in all cases.
The conclusion drawn from these results is that 7th day visceral endoderm from
both regions of the egg cylinder has the inherent ability to synthesize AFP, but
that synthesis is inhibited by a contact-associated interaction with EX ectoderm.
It appears that this interaction can inhibit AFP synthesis after it has already
been initiated in the VE endoderm, and that contact must be continuous to
maintain inhibition in the visceral endoderm. The few cases where low levels
of AFP were still observed after association could still be due to later establishment of contact and delayed inhibition.
Initiation of AFP synthesis in isolated visceral endoderm
It is conceivable that the inhibition of AFP synthesis in visceral endoderm
cells in vivo becomes stabilized after a prolonged interaction with EX ectoderm,
such that maintenance of the interaction is no longer necessary. To test whether
this is the case, the capacity for AFP synthesis in the visceral endoderm of 8th
day embryos was compared to that in 7th day embryos.
VE and VEX endoderm tissues were isolated enzymically from both 7th
and 8th day embryos and cultured under identical conditions to compare the
onset and progression of AFP synthesis. Embryos on the 7th day ranged from
pre-primitive streak to early primitive streak stages of development, while
8th day embryos were advanced egg cylinders with large embryonic cavities.
Tissues were fixed for the immunoperoxidase reaction after 0, 6, 12, 24, 48 and
72 h in culture. The results are summarized in Table 2.
AFP was present in VE endoderm cells from both 7th and 8th day embryos
immediately after isolation and at all stages during the culture period. AFP was
first observed in isolated VEX endoderm cells after 12 h in culture. The degree
of peroxidase staining was similar in VEX endoderm tissue from both 7th and
8th day embryos. After 12 h low levels were present, which increased to very
high levels by 24 h. These results show that 8th day VEX endoderm does not
differ from the 7th day VEX endoderm in capacity for AFP synthesis, and hence
no stable inhibition of AFP synthesis has taken place over this time interval.
However, unlike the VE endoderm in which most cells were AFP-positive after
12 h in culture, patches of AFP-negative cells were often observed in the VEX
endoderm cultures even after 72 h. This could be due to contamination by a
few parietal endoderm cells which form a layer continuous with the VEX endoderm in the intact embryo but do not synthesize AFP (Dziadek & Adamson,
1978).
The results show that the interaction between EX ectoderm and visceral
endoderm tissues does not induce a stable change in the capacity for AFP
10
EMB +6
142
M. DZIADEK
VE
VEX
50//m
3A
50 //m
3B
EX
VEX
MG
50/im
4B
4A
MG
50//m
Figs. 3-5. Immunoperoxidase reaction on sections of ectoderm and visceral endoderm aggregates after 48 h in culture.
Fig. 3. VE (A) and VEX (B) endoderm cells associated with E ectoderm tissue
contain AFP.
Fig. 4. VE (A) and VEX (B) endoderm cells associated with EX ectoderm tissue do
not contain AFP, but some outer cells contain melanin granules.
Fig. 5. An aggregate where VE endoderm cells remained as a clump attached to EX
ectoderm tissue. Cells closely associated with the EX ectoderm do not contain AFP
but are labelled with melanin granules, and cells in the adhering clump are AFPpositive. E, embryonic ectoderm; EX, extra-embryonic ectoderm; VE, visceral
embryonic endoderm; VEX, visceral extra-embryonic endoderm; MG, melanin
granules.
Modulation of AFP synthesis
143
Table 2. The presence ( + ) or absence ( —) of AFP in isolated visceral endoderm.
tissue at various time intervals between 0 and 72 h in culture
The number of observations are indicated in parentheses.
Oh
7th
8th
7th
8th
day,
day,
day,
day,
VE endoderm
VE endoderm
VEX endoderm
VEX endoderm
+
+
-
(4)
(14)
(4)
(18)
6h
+ (4)
+ (4)
- (4)
-(4)
12 h
+
+
+
+
(9)
(13)
(7)
(14)
24 h
+ (8)
+ 02)
+ (9)
+ (12)
48 h
+
+
+
+
(15)
(14)
(18)
(23)
72 h
+
+
+
+
(7)
(14)
(8)
(10)
synthesis by the visceral endoderm, but has a secondary modulatory role in the
inhibition of phenotypic expression. Release from inhibition occurs when
visceral endoderm is isolated from EX ectoderm.
DISCUSSION
In a study of the localization and synthesis of AFP during early mouse
embryogenesis it was observed that the AFP-positive and AFP-negative cell
subpopulations of the visceral endoderm were closely associated with the type
of underlying ectoderm tissue (Dziadek & Adamson, 1978). The question arose
whether tissue interactions were involved in the synthetic activities of the
visceral endoderm. The present study establishes that this is the case. The results
show that VE and VEX endoderm cells from both 7th and 8th day egg cylinders
can synthesize AFP when growing in isolation from other embryonic tissues.
Modulation of AFP synthesis occurs through an inhibitory interaction with EX
ectoderm tissue. The two subpopulations of the visceral endoderm therefore do
not differ in their ability to synthesize AFP, but in the phenotypic expression of
this ability. Initiation of AFP synthesis in VEX endoderm cells after release
from the influence of EX ectoderm tissue follows a similar pattern in cells from
both 7th and 8th day embryos, which suggests that the mechanism for inhibition
of AFP synthesis may be the same over this developmental period.
A major criticism of studies involving tissue interactions in vitro concerns
the influence of culture conditions on phenotypic expression, as well as the
possible influence of enzymes used for tissue separation. However, in the
interaction studies reported here inhibition does not appear to be due to any
deficiencies of the culture conditions. In every experiment, visceral endoderm
cells were observed to synthesize AFP except when in direct contact with EX
ectoderm. Inhibition is not due to any factors in the culture medium which are
released by EX ectoderm, since visceral endoderm cells growing in the same
culture drop but not associated with EX ectoderm were observed to contain
AFP. Very little cell degeneration was obvious during tissue culture, but even if
degradation products were accumulating in the medium, these were not likely
to be causing the inhibition observed for the same reason as above. It is con-
144
M. DZIADEK
eluded that the absence of AFP in visceral endoderm cells which are associated
with EX ectoderm tissue, both in vivo and in vitro, is due to an inhibitory
influence of the EX ectoderm.
The results presented in this report show that visceral endoderm cells can
change their phenotype in vitro when the tissue associations normally present
in vivo are altered. Whether such changes in phenotype occur in visceral endoderm cells during the development of the intact embryo is not clear, since the
movement of cells in the visceral endoderm layer with respect to underlying
tissues has not been studied. Observations on the relationship between phenotypic expression and the type of tissue association in the in vitro experiments
reported here correspond to the observations on intact 7th and 8th day embryos
(Dziadek & Adamson, 1978). However, in 9th day embryos the position of
AFP-positive and AFP-negative visceral endoderm cells does not correlate
precisely with the type of underlying tissue. Visceral endoderm cells which are
situated around the exocoelomic cavity now overlie a layer of extra-embryonic
mesoderm cells. Only a small proportion of these visceral endoderm cells contain AFP, and these are positioned in a continuous band around the midgirth of
the embryo (Dziadek & Adamson, 1978). From the results presentedinthis report
one would expect all these cells to be AFP-positive, since they are not in contact
with EX ectoderm tissue. It seems reasonable to propose that AFP-negative
visceral endoderm cells in this region have moved away from the influence of
the EX ectoderm but have not yet initiated AFP synthesis. Migration of extraembryonic mesoderm into the exocoelomic cavity and subsequent expansion
of this cavity could create a change in tissue associations. From the observations
of initiation of AFP synthesis in vitro, cells which have moved from the influence
of EX ectoderm should synthesize low levels of AFP after 12 h. On the 10th day
of development all visceral endoderm cells of the yolk sac contain high levels
of AFP (Dziadek & Adamson, 1978), which substantiates such a proposal.
However, this question needs further investigation.
The tissue interaction reported in this study occurs at the stage in mouse
development when only two germ layers are present, and is the first example of
modulation of phenotypic expression in the early mouse embryo through an
inhibitory influence. However, an earlier tissue interaction is thought to occur
between the inner cell mass and trophectoderm of blastocysts (Gardner, 1971,
1972; Ansell & Snow, 1974; Gardner & Johnson, 1975). The different influences
of the E and EX ectoderm is not surprising, since they have different origins in
the early embryo (Gardner & Johnson, 1975; Gardner & Papaioannou, 1975),
and show different properties when grown in vitro and in ectopic sites (Diwan &
Stevens 1975; Rossant& Ofer, 1977). It remains to be determined which properties
of the EX ectoderm are involved in the inhibition of AFP synthesis in the overlying visceral endoderm.
The mechanisms which control AFP synthesis in the visceral endoderm of
the early embryo may be similar to controls of AFP synthesis in other tissues.
Modulation of AFP synthesis
145
The second major site of AFP synthesis during embryogenesis is the foetal liver
(Engelhardt et al. 1971; Abelev, 1971; Wilson & Zimmerman, 1976). The
developmental mechanisms by which transitory foetal antigens such as AFP
become inhibited after birth are not yet understood. Evidence that inhibition is
not completely irreversible comes from studies showing the reappearance of
AFP synthesis in mice with hepatomas (Abelev, 1971; Engelhardt et al. 1971)
and during liver regeneration (Engelhardt et al. 1976). Studies on the controls
of AFP synthesis during embryogenesis and the maintenance of these controls
during foetal and adult life may provide a basis for studies on the regulation of
gene expression in carcinogenesis.
I wish to thank Dr Chris Graham, Dr Eileen Adamson and Ms Jolanta Opas for critical
reading of the manuscript. I am supported by a Flinders University of South Australia
Overseas Scholarship.
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