/ . Embryo!, exp. Morph. Vol. 61, pp. 103-116, 1981
Printed in Great Britain © Company of Biologists Limited 1981
103
Molecular studies on cells of
the trophectodermal lineage of the
postimplantation mouse
embryo
By M. H. JOHNSON 1 AND J. ROSSANT 2
From the Department of Anatomy, University of Cambridge
SUMMARY
Embryonic ectoderm (EmE), extraembryonic ectoderm (EE), ectoplacental cone diploid
cells (EPC) and secondary giant cells (GC) were isolated from 7^-day mouse embryos and
their polypeptide synthetic profile assessed by fluorography of 2D polyacrylamide gels.
Fifty polypeptides showed different distributions amongst the tissues, permitting characterization of each tissue by an array of polypeptide marken; typical for the tissue at that
developmental stage. The three tissues on the presumptive trophectoderm lineage did not
show identical synthetic patterns. However, culture of EE cells in vitro resulted in conversion
of their polypeptide synthetic profile to that of EPC after 2 days and of GC after 6 days,
whilst culture of EPC cells converted their polypeptide synthetic profile to that of GC after
only 4 days. These changes in polypeptide synthesis correlated well with the ploidy levels
of the tissues at different times in culture.
INTRODUCTION
The analysis of cell lineage and prospective fate in development requires
the manipulation and analysis of marked populations of cells. Using such
approaches it has proved possible to draw up a tentative fate map of the early
mouse embryo (see Gardner & Johnson, 1975; Gardner & Papaioannou, 1975;
Rossant, 1977; Rossant & Papaioannou, 1977). It is believed that the ICM
of the blastocyst gives rise to the primitive ectoderm (which later forms the
definitive ectoderm, mesoderm and endoderm of the foetus) and primitive
endoderm of the postimplantation embryo. The trophectoderm of the blastocyst
forms the ectoplacental cone and primary and secondary giant cells. There is
also evidence to suggest that the extraembryonic ectoderm, which later forms
the ectoderm of the chorion, arises from the trophectoderm (Gardner,
Papaioannou & Barton, 1973; Gardner & Johnson, 1973, 1975; Rossant &
1
Author's address: Department of Anatomy, Downing Street, Cambridge CB2 3DY, U.K.
Author's address: Department of Biological Sciences, Brock University, Glenridge
Campus, St Catharine's, Ontario L2S 3A1, Canada.
2
104
M. H. JOHNSON AND J. ROSSANT
Giant cells
(GO
Diploid
ectoplacental cone
(EPC)
Extraembryonic
ectoderm
(EE)
Exocoelom
Amnion
Embryonic
ectoderm
(EmE)
Mesoderm
.Endoderm
Fig. 1. Diagram of 7^-day mouse embryo. The dotted lines mark the lines of
dissection.
Ofer, 1977; Frels, Rossant and Chapman, 1979) and not from the ICM as
has been suggested by some authors (Robinson, 1904; Rugh, 1968).
We present here data obtained in an attempt to analyse at the molecular
level the relationships between tissues of the implanted embryo. The polypeptides synthesized by embryonic ectoderm (EmE), extraembryonic ectoderm
(EE), ectoplacental cone diploid tissue (EPC) and secondary giant cells (GC)
have been analysed (a) to determine whether tissue-specific markers exist
and (b) to discover whether associations of lineage and fate between constituent
cell populations can be established on the basis of molecular identity. EPC
and EE were also cultured in vitro and their polypeptide synthetic profiles
analysed during the process of transformation to GCs (Rossant & Ofer, 1977).
MATERIALS AND METHODS
Recovery of embryos, dissection and incubation
Random-bred mice of either CFLP (Hacking & Churchill Ltd) or PO
(Pathology, Oxford) stock were used throughout this study. Previous studies
have not revealed any consistent differences between preimplantation embryos
from different mouse strains (Levinson, Goodfellow, Vadeboncoeur &
Molecular studies on trophectodermal-lineage cells of mouse
105
McDevitt, 1978; Van Blerkom & Johnson, unpublished). Phosphate-buffered
medium (Whittingham & Wales, 1969) + 10% heat-inactivated foetal calf
serum was used for dissection and storage of embryos. Embryos were dissected
from the uteri of mice on the 8th day of pregnancy (7^-day embryos). At this
stage, the primitive streak is well established and the amniotic folds are almost
complete, separating the EE from the embryonic shield. Using fine watchmaker's forceps, Reichert's membrane was torn away carefully, to avoid
damaging the overlying secondary GCs. A cut was then made close to the
origin of Reichert's membrane to separate the embryonic and extraembryonic
regions from the EPC. A solid lump of presumed diploid trophoblast cells can
be seen in the core of the EPC, and this was dissected out as cleanly as possible
from the overlying cells and maternal blood cells. These remaining fragments
were retained for separation of GCs. The egg cylinders were cut into extraembryonic, exocoelomic and embryonic regions with glass needles (Fig. 1).
The exocoelomic fragments were discarded.
Separation of the germ layers in the embryonic and extraembryonic
fragments was achieved by incubating in 2-5 % Pancreatin and 0-5 % trypsin
in Ca-, Mg-free Tyrode's saline at 4°C for 10-20 min, as described previously
(Rossant & Ofer, 1977). After sucking up and down in flame-polished
micropipettes, the EE can be separated cleanly from the endoderm and
mesoderm and the EmE can be separated from the endoderm and most of the
mesoderm except in the region of the primitive streak. This region was cut
away and discarded so that only pure EmE tissue was retained. The presumptive
diploid EPC fragments were also incubated in the Pancreatin/trypsin mixture
for 10 min. This procedure clears any remaining intercellular matrix, damaged
cells and contaminating maternal cells from the ectoplacental lumps. To obtain
clean preparations of secondary GCs, the fragments of GCs, Reichert's
membrane and adhering maternal cells were incubated in 0-25 % trypsin in
Ca-, Mg-free saline at 37 °C for 20 min. After sucking' up and down in
progressively smaller micropipettes, giant cells were freed from other tissues
and could be sucked up and collected in groups of at least 50 in fresh medium.
For analysis of immediate protein synthesis, individual EPCs, EEs, EmEs
and groups of secondary GCs were incubated in 0-1 ml RPMI (Flow Labs)
containing 10 |il [S35]methionine (Radiochemicals, Amersham; specific activity
approx. 1000 Ci/mmol) at 37 °C in 5 % CO2, 90 % N 2 and 5 % O 2 for 4 h.
After this time the medium was withdrawn and the samples lysed in c. 30 ul of
2D buffer (O'Farrell, 1975), and stored at -70°C.
EEs and EPCs were also cultured in RPMI + 10 % FCS for periods of up to
6 days, by which time both fragments transformed into giant cells (Rossant &
Ofer, 1977). In this case, groups of three fragments were cultured together and
pooled for analysis. Incubation in the labelled amino acid was performed as
above after 2, 4 and 6 days of culture.
106
M. H. JOHNSON AND J. ROSSANT
2C
4C
8C
16C
10
50
2C
1
20 r-
100
4C
150
200
250
Absorbance units
ml
300
h if
350
400
3f nuclei
o
15
Jl
d
Z
5 -
r
,
1
,'—i
50
100
Absorbance units
150
Fig. 2. Histograms of DNA contents of (A) secondary giant-cell nuclei, (B) diploid
ectoplacental cone nuclei. C values are derived by comparison with and extrapolation from the absorbance readings of control liver nuclei. Data from three
different samples stained identically, were pooled for each histogram. The diploid
ectoplacental cone samples contained cells with 2C and 4C DNA levels but no higher
C values were found. Quite a large proportion of the cells appear to be tetraploid
rather than diploid but this may simply reflect the fact that this cell population is
highly proliferative and many cells will be in the tetraploid G2 phase of the cell
cycle. DNA values for the secondary giant-cell samples reveal a much wider spread
but few, if any, cells appear to be diploid. Most cells appear to be around the
8C level but some higher values were also observed. Thus, the isolation techniques
for diploid ectoplacental cone and secondary giant cells allow very little crosscontamination between the two cell types and so comparison of their electrophoretic profiles is valid.
Microdensitometry
Cell spreads were prepared from EPC and GC isolated directly from the
embryos, and from EPC and EE, after various times in culture. A mouse liver
imprint was also placed on each slide and all the cells were fixed in acetic
alcohol. The cell spreads were stained by the Feulgen technique for DNA
(Pearse, 1972). Microdensitometry measurements were made using a Leitz
MPV microspectrophotometer, with the interference filter set to give peak
transmission at 550 nm. Control liver readings were made for each slide. The
results were expressed in the form of histograms of total absorbance measured
in arbitrary units. These histograms were then calibrated in multiples of the
haploid DNA values (C) by comparison with and extrapolation from the liver
Molecular studies on trophecto dermal-lineage cells of mouse
107
controls, whose cells contain 2C, 4C and 8C values of DNA. The histograms
of EPC and GC isolated directly from the embryo show little overlap in DNA
values between the two tissues (Fig. 2), indicating satisfactory isolation methods.
Electrophoresis
Electrophoretic separation of polypeptides was performed on twodimensional gels as described by O'Farrell (1975). Samples for two-dimensional
analysis were thawed and frozen gently 3X, urea crystals were added to
saturation, and 20 ul of sample were applied to preequilibrated cylindrical
4 % acrylamide gels (O'Farrell, 1975). Samples were run for a total of 6000 volt
hours, the last hour of which was at 800 V. Gels were then equilibrated with
two changes of SDS sample buffer for 1^ h and applied to the top of gradient
slab gels (7-15% acrylamide 110x150 mm overlain by 4-5% acrylamide
10 x 150 mm) embedded in 1 % agarose in double-strength SDS buffer. The
samples were run at 15mamps for 4-5 h. Gels were fixed in 25% TCA,
processed through 7 % acetic acid, three changes of DMSO (2 h each), 20 % PPO
in DMSO (2 h), water (three rinses), 7 % acetic acid (2 h) and then dried onto
cards under vacuum (Bonner & Laskey, 1974). Dried gels were exposed to
preflashed Fuji X-ray film (Laskey & Mills, 1975) for 1 to 8 weeks at - 7 0 °C.
Films were developed for 4 min at 20 °C in Kodak DX-80 Developer. Several
films of varying exposure times were available for each preparation. The film
used for the analysis was selected by examination of several reference polypeptides for roughly equivalent intensity in each film (see below).
Procedure for gel analysis
The films produced by exposure to gels were viewed on a modified X-ray
viewer. A film of an EmE sample was selected as standard, and compared first
with all other films from EmE separations. Most polypeptides migrated in
similar relative positions and were of similar relative intensities. These polypeptides were noted and were used in compaiisons with films from other
tissues. A few polypeptides were variable between gels. Some of this variation
appeared due to secondary modification of invariant polypeptides, but in
a few cases polypeptides appeared in one or two gels but no others. In
subsequent comparisons with other gels, these polypeptides were either not
detected or were detected variably in these tissues also.
Sufficient similarities existed between films from different tissues for a set
of reference polypeptides, common to all films, to be established. These were
used first to assess the degree of any distortions of gels during processing, and
then as the reference points for adjacent tissue-limited polypeptides. All comparisons between gels were made on four separate occasions at least 2 weeks
apart and the results from each analysis recorded and compared subsequently.
Only polypeptides which were consistently scored in the same relative position
and intensity for each gel were considered to be reliable markers.
108
M. H. JOHNSON AND J. ROSSANT
(O
Molecular studies on trophectodermal-lineage cells of mouse
109
A total of 36 gels was examined (7 EmE, 6 EE, 15 EPC and 8 GC) and
50 polypeptides were found that showed consistent differences. Each of the
50 polypeptides examined in every gel was evaluated as strong, weak or not
detectable relative to the reference polypeptides or as not scorable due to
streaking or other local gel imperfections. When this analysis was completed,
a further comparison was made with eight 2D separations of polypeptides from
EPC tissue culture in vitro for 2 or 4 days and twelve from EE tissue cultured
in vitro for 2, 4 or 6 days.
Each of the 50 polypeptide spots was assigned a reference code. Those
polypeptides which showed a migration identical to polypeptides studied
previously on preimplantation-stage embryos were assigned the same code
number as used previously (Handyside & Johnson, 1978; Johnson, 1979).
Other variant polypeptides were assigned a letter code. Certain of the polypeptides (codes N, h, i and 14) appear to be present as two or three spots often
along a horizontal (isoelectric focusing) axis and show reciprocal changes in
intensity. In the figures and tables, the components of these groups of polypeptides are given suffices 1 and 2 or 1,2 and 3 reading position from basic
to acidic. On each gel a small black or white point marks the position of the
trailing (left) edge of the polypeptide or its position and the adjacent number/
letter code is above or to the left of the point.
For the block summary diagrams, each square represents an aggregate score
of all the gel separation patterns examined for the polypeptide and tissue in
question.
RESULTS
Figures 3-6 show representative plates derived from two-dimensional
electrophoretic analysis of EmE, EE, EPC and GC. A summary of the distribution of each of the tissue-marker polypeptides amongst the 36 films analysed
is presented in Fig. 7 and Table 1. Table 2 summarizes the extent to which
different tissues show molecular identity in regard to their synthesis of the
tissue-marker polypeptides.
Two-dimensional electrophoresis was performed on samples of both EPC
and EE which had been cultured in vitro for 2-6 days. During this period, both
cell types transform their appearance (Rossant and Ofer, 1977) and DNA
content to resemble GCs. However, the.time course of transformation was
different in the two tissues (Fig. 8). EE cells remained diploid for at least 2 days
FIGURES
3-6
Fluorographs of representative two-dimensional gels of polypeptides derived from
7-1-day EmE (Fig. 3), EE (Fig. 4), EPC (Fig. 5) and GC (Fig. 6). Individual polypeptides are marked with a white or black spot to the left of their position and
assigned a white or black reference code above or to the left. (White and black
are used for maximum contrast only and have no other significance). Fig. 3 indicates
pH and M.W. (x 104) range of separations.
110
M. H. JOHNSON AND J. ROSSANT
Table 1. Code references ofpolypeptides limited to any tissue or group of tissues
1. Embryonic ectoderm only
2. Embryonic ectoderm + extraembryonic ectoderm
- f Extraembryonic ectoderm + ectoplacental cone
DeGUab
K S T W 11 h3
N 2 9 i2
' \ Extraembryonic ectoderm + ectoplacental cone + giant cells
. | Ectoplacental cone
FL2VYZfl5i1
h2
" 1 Ectoplacental cone + giant cells
AC413OPQ7adhx
5. Giant cells
B 14X 7b
6. Embryonic ectoderm + extraembryonic ectoderm + ectoplacental cone E 1 4 2 H I J R X
Table 2. Total number of polypeptides which show identical presence / absence
patterns* in gel separation of different tissues ( + ve association in brackets)
EmE
EE
EPC
GC
EmE
EE
EPC
GC
46 (20)
27 (13)
46 (25)
10(7)
27 (18)
46 (30)
6(2)
17 (10)
31 (20)
46 (24)
* Differences in overall intensity have been ignored in this comparison; thus polypeptides
142, M, 3 and 16 are not included.
in culture before beginning to endoreduplicate whereas EPC cells are already
moving away from diploid DNA values by 2 days. Examination of the films
derived from 2D separations shows a corresponding molecular transformation
to a secondary giant-cell pattern. The results of these separations are
summarized in Fig. 9, which show that (a) the final molecular pattern for both
tissues is almost identical to that of secondary giant cells (b) EE takes approximately 2 days longer than EPC to transform totally its synthetic pattern (c) the
earliest molecular transformations shown by EE involve primarily those
polypeptides by which extraembryonic ectoderm differs from EPC (polypeptides A C E K L M 3 4 16 1 3 O P Q 7 a l l Z d f g l 5 h 1 h 2 h 3 i 1 i 2 - only
polypeptides S T X N j and W do not conform to this pattern). This observation
suggests that EE transforms to GC through an EPC type profile, a conclusion
confirmed by polypeptides g, h 2 and ix which are weak in EE, present in EPC
and weak or absent in GC. During culture of EE in vitro these three polypeptides show a transitory increase followed by a decline.
DISCUSSION
Examination of the two-dimensional electrophoretic profiles of polypeptides
synthesized by various tissues of the 7^-day mouse embryo has revealed several
polypeptides that appear to be limited to one tissue or group of tissues (Table 1).
Molecular studies on trophectodermal-lineage cells of mouse
A B C D E e
Q
9 R S T U V
F G 14 t 14 2 H
I
J
K L 2 M 3 4
W 7a 7b X Y II Z
a
b
d
f
P
g 15 h, h 2 h 3 \x i2
^ | Strongly detectable
Weakly detectable
f
Not detectable
j Trace detectable
N,N 2 16 13 0
111
Fig. 7. Summary of polypeptide synthetic profiles of 7^-day embryonic ectoderm
(EmE), extraembryonic ectoderm (EE), ectoplacental cone cells (EPC) and
secondary giant cells (GC).
Each of the four tissues analysed is characterized by a typical array of
endogenous polypeptide markers, detection of which as a group may be
taken as diagnostic of the tissue at that point in its development. However,
although this analysis may prove to have been useful in providing a complex
of stage and tissue-specific markers, it does not provide clear and independent
support at the molecular level for experimentally-derived cell-lineage relationships. Thus, as may be seen from Table 2, EE appears to occupy a position
roughly midway between EmE and EPC in its communality of polypeptides.
Such a result is not surprising for both technical and theoretical reasons.
First, the gel patterns observed after labelling and two-dimensional electrophoresis of embryonic polypeptides are the result of a complex sequence of
events. The final gel patterns may be influenced by differences in the developmental staging of equivalent age tissues, slight differences in techniques of
isolation and handling of tissues from experiment to experiment, possibility
of viral or bacterial contamination of tissues during handling and culture,
accessibility to and toxicity for the tissues by the label, responses of the tissue
to isolation and short-term culture and post-translational modification of
polypeptides in situ and during processing. All of these features might operate
differently in different tissues. Whilst it is highly unlikely that all the differences
between tissues are being induced experimentally, each tissue in fact synthesizing identical polypeptides in situ, it is not improbable that some of the
differences observed are due to factors such as these. The problem posed by
the technical artifacts can be reduced somewhat by standardizing conditions
and their magnitude can be assessed by co-labelling and co-processing of
different tissues. However, for tissues of such complexity, it will not be easy
112
M. H. JOHNSON AND J. ROSSANT
8C
2C
4C
I
20 r
I
w\
15
EE
2d
10
n
100
50
2C
15
5
73 10
3
C
6
-
5 -
1
EE
r
10
-
4d
n
u 4_i n
L
50
10
8C
1
n
o
Z
4C
1
100
1
—, n
150
200
2C
4C
8C
I
1
1
5
J
m r
50
P-
250
nn
1
fL-, rLrjJ~~L|
250
300
n 250
300
150
200
Absorbance units
2C
.
300
16 C
n
100
i—i
EE
6d
1
n
n
350
8C
I
epc
2d
50
2. 15
100
150
200
2C
4C
8C
16C
1
1
1
1
lOi
epc
4d
r
5 -
n
n_
LJ
i
50
100
1
150
200
Absorbance units
n
250
i
300
r-\
n
350
Molecular studies on trophectodermal-lineage cells of mouse
113
A C 14, L 3 4 16 13 O P Q 7a 7b Y Z d 15 h,
I I I I II
EE
EE
EPC
EE 4
EPC 2
EE 6
EPC 4
GC 0
F
1£E
EE 2
EPC 0
EE 4
EPC 2
EE
E
H
1 J
K N, R
II
f
h , i, M
9
N, W
0
•
•
6
EPC 4
GC
0
Fig. 9. Summary of polypeptide synthetic profiles of 7|-day extraembryonic
ectoderm immediately after isolation (EE 0), after 2, 4 and 6 days in culture
(EE2, EE4 and EE 6) and of 7^-day ectoplacental cone cells immediately after
isolation (EPC 0), after 2 and 4 days in culture (EPC 2 and EPC 4) and of secondary
giant cells immediately after isolation (GC 0).
to eliminate these problems entirely. In this study, only a minority of the
polypeptides being synthesized by the tissues are examined, the rest being either
excluded from the isoelectric range analysed or too weak for detection, and
of this minority, most are present in all films, some show a variable and
apparently random appearance and only a group of 50 show consistent tissue
Fig. 8. Histograms of DNA contents of EE and EPC nuclei after various times in
culture. These histograms were prepared in the same way as those in Fig. 2. EE cells
remain diploid for 2 days before transforming into giant cells whereas the peak
DNA values for EPC are already greater than 2C by 2 days. The histograms of
EE at 4 days and EPC at 2 days are almost exactly superimposable, as are the
histograms for EE at 6 days and EPC at 4 days. Tn the later cultures, the largest
DNA values are underestimates because the largest cells tended to be disrupted
during preparation of the cell spreads.
114
M. H. JOHNSON AND J. ROSSANT
patterns. This highly selected minority of polypeptides thus constitute a reliable
set of markers for a tissue taken at a defined age and processed in a defined way.
They do not represent the complete pattern of distinctive polypeptides
synthesized in situ. Their value is primarily diagnostic rather than analytic
(see also Dewey, Fuller & Mintz, 1978, for a discussion of this problem).
Second, even assuming that the gel patterns observed did represent the
synthetic pattern in situ, there would be no particular reason to expect that
the synthesis of those polypeptides which reflect a particular tissue's past
lineage or future potential should predominate over synthesis of those which
are characteristic of its present differentiated state. Thus, although experimental
studies strongly support a common lineage and potential for EE and EPC
(Gardner & Papaioannou, 1975), the tissue organization of the 7-^-day EE into
an epithelium resembles more closely the differentiated state of EmE than EPC.
The polypeptide synthetic profile of EE not surprisingly reflects all of these
features of its organization. General molecular comparisons of this sort seem
unlikely to shed light on cell lineages.
Considerations such as these make it unprofitable to compare in any detail
the results described in this paper with earlier molecular studies described
previously for morulae, blastocysts and teratocarcinoma cells (Van Blerkom,
Barton & Johnson, 1976; Handyside & Johnson, 1978; Dewey et al. 1978).
Although the polypeptides characterized previously as trophectodermal
markers (2, 3, 4, 7, 13 and 16) were detected in this study only in cells of the
putative trophectodermal lineage and thus appear to conform to the putative
lineage, of the three polypeptides detected in this study that co-migrate with
previously described 1CM marker polypeptides, number 11 is present in both
EmE and EE, number 15 is absent only from EmE and number 14 is synthesized
by all four tissues. For the reasons outlined above, and given the increased
embryonic complexity that has occurred over the 4-day period separating the
two embryonic stages analysed, little significance can be placed on this
observation.
A more profitable approach to molecular studies of lineage and potential
comes from the analysis of the changes in molecular expression with time by
a tissue exposed to altered conditions. For example, we have previously
demonstrated that groups of inside cells isolated from morulae and blastocysts
show, on their isolation and culture in vitro, a molecular transition towards
a blastocyst-type pattern. The completeness of this altered molecular response
can be correlated with the pluripotentiality of the inside cells (Johnson, 1979).
Using a comparable approach with isolated and cultured EPC and EE,
a striking correlation between transformation at molecular and morphological
levels has been observed. Thus, the EE cells in culture transform over the
first 2 days to an EPC-type pattern during which period they remain diploid.
Then over a further 4 days both EE and EPC transform together to a GC-type
polypeptide pattern and both become polyploid (Figs. 8 and 9). Only 7 of 42
Molecular studies on trophectodermal-lineage cells of mouse
115
polypeptides (B, X, N l 5 S, T, V and W) do not conform strictly to this sequence,
which is remarkable considering that the gels represent static sampling of
a continuous transition. Thus, trophoblast giant cells generated in vivo or
in vitro show identical polypeptide synthetic profiles whatever their origin,
indicating a common pathway of differentiation open to all trophectoderm
derivatives and raising the possibility that in the normal embryo the EE cells
may act as a reservoir of cells that can be recruited to the EPC to generate
new giant cells.
We would like to acknowledge the technical assistance of Debbie Eager. This work was
supported by grants to M.H.J. from the MRC and the Ford Foundation. J.R. was a Beit
Memorial Fellow and is now supported by the Canadian NSERC. We wish to thank Dr
Y. Masui, Department of Zoology, University of Toronto for use of the microspectrophotometer.
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(Received 17 December 1979, revised 16 August 1980)