/ . EmbryoL exp. Morph. Vol. 49, pp. 277-294, 1979
Printed in Great Britain © Company of Biologists Limited 1979
277
Formation and consequences of cell patterns in
preimplantation mouse development
By C. F. GRAHAM 1 AND E. LEHTONEN 1
From the Zoology Department, University of Oxford
SUMMARY
The behaviour of groups of cells was studied in culture during preimplantation mouse
development. The following observations were made with intact embryos minus the zona
pellucida and with embryos whose cells had been dissociated and recombined. The form of
the 4-cell embryo was related to the behaviour of the first cell to divide to this stage. The form
of the 8-cell embryo depended on contact between the groups of four cells each derived from
a single cell at the 2-cell stage. The form of the 16-cell embryo depended on cell movement
during and after division from the 8- to the 16-cell stage. These results suggest that the morphogenetic movements of these early embryonic cells are principally governed by continuous cell
interactions after fertilization. The cell surfaces of the embryos were examined with scanning
electron microscopy in an attempt to discriminate between the mechanisms which could
account for these movements.
INTRODUCTION
We attempt to show that the behaviour of cells during the early cleavage
stages of the mouse development depends on continuous cell interactions. These
cell interactions appear to account for many features of the mouse cell lineage
and in particular for the allocation of cells to the inner cell mass (ICM) and to
the trophectoderm of the blastocyst.
The cell lineage of the preimplantation mouse embryo has several regular
features (Lewis & Wright, 1935; Wilson, Bolton & Cuttler, 1972; Graham &
Deussen, 1978; Kelly, Mulnard & Graham, 1978). Inside the zona pellucida, all
cells of the 4-cell embryo usually touch all other cells and each has the same
number of cell contacts. At the 8-cell stage, cells have different numbers of cell
contacts and the frequency with which they contribute daughter cells to the
ICM is related to the number of cell contacts. We attempt to explain the mechanism by which the 8-cell-stage pattern is set up and has its effects on cell
allocation.
1
Authors'1 address: Zoology Deparment, South Parks Road, Oxford, 0X1 3PS, U.K.
278
C. F. GRAHAM AND E. LEHTONEN
MATERIALS AND METHODS
Supply and culture of embryos. The embryos were from natural matings and
were A2G, C3H, and (C57BL6 x CBA) F 2 and CFLP outbred stock (Anglia
Laboratory Animals Ltd.). They were dissected into pre-warmed and preequilibrated Whitten's medium (1971). The zona pellucida was partially dissolved with pronase and usually pipetted off each embryo (Calbiochem Co.,
U.K., technique of Mintz, 1967). The embryos were cultured on bacteriological
dishes (Sterilin Ltd., Richmond, Surrey, U.K.), in microdrops (approximate
volume 0-05 ml) under paraffin oil (selected for absence of toxicity to cultured
embryos, Boots Pure Drug Co., U.K.) in a humidified gas mixture of 5 % CO2,
5 % O2, and 90 % N 2 at 37 °C.
The embryos were drawn and photographed on an inverted microscope with
a channelled microscope stage warmed with water from a thermocirculator
(Churchill Ltd., Greenford, Middx., U.K.). The embryos were regularly observed
at 20 min intervals during the periods when cells were expected to divide, and
they were also continuously observed if a cell was seen to enter cytokinesis at
one of these observation intervals.
In some experiments, a single cell was lysed inside the zona contained 2-cell
embryo using a needle held on Leitz micromanipulator (Graham & Deussen,
1978).
Dissociation of embryos. Embryos were dissociated after pronase treatment
by culture for 15 min in Whitten's medium in which the calcium concentration
had been lowered to 0-02 HIM. They were blown apart by pipetting through a
flame polished micropipette with a mouthpiece. They were immediately replaced
in normal Whitten's medium after dissociation. The low calcium medium was
not required for dissociation at the 2-cell stage.
Culture as monolayers. 8-cell embryos were dissociated into two groups of
four cells by the above procedure. Each four-cell group was placed in a large drop
(approximate diameter 5 mm) under liquid paraffin. The medium was withdrawn
so that the medium/paraffin oil boundary was in contact with the cells and held
them flat against the bottom of the culture dish. This procedure was necessary
to stop the cells moving on top of each other and obscuring cell outlines. This
procedure did not kill the cells if the four-cell groups were returned to a large
drop after less than 10 h in these conditions; about 90% formed half sized
blastocysts.
Fixations'. The embryos were fixed in Heidenhain's fixative (Tarkowski &
Wroblewska, 1967). After fixation the embryos were pipetted into a warm agar
solution (1 % w/v) in 6 % NaCl (w/v) on a slide lying on a hot plate. When the
agar had set, a block containing the embryos was cut out. It was embedded in
paraffin wax and cut at 7 /*m. In some cases the embryos were fixed, embedded
in araldite, and sectioned at 1 /*m as described previously (Graham & Deussen,
1978).
Cell patterns in mouse development
279
The wax embedded sections were stained in Mayer's haemalum and light
green. The plastic embedded sections were stained with hot toluidine blue, as
described previously (Graham & Deussen, 1978).
Scanning electron microscopy: For scanning electron microscopy (SEM), the
zona pellucida was partially digested with pronase and then pipetted off the
embryo in Whitten's medium. Next the embryos were cultured in this medium
for at least 60 min. These embryos and cell arrangements from the other experiments were briefly rinsed in Whitten's medium without albumin and routinely
fixed in 2-5 % (v/v) glutaraldehyde in 0-1 M cacodylate buffer with 2 mM-CaCl2
at pH 7-4 for 60 min at room temperature. Various combinations of glutaraldehyde, paraformaldehyde, and acrolein in either cacodylate or phosphate buffer
or in Whitten's medium without albumin gave identical results; variation in
temperature during fixation (from + 4 °C to + 37 °C) had no visible effect on the
surface morphology of the cells.
After fixation, the cell arrangements and embryos were attached to the concave surface of coverslip fragments which had been curved in the flame of a gas
microburner. These fragments were either treated with 0-1 % (w/v) poly-1-lysine
in water (McKeehan & Ham, 1976) and the cells stuck when pipetted onto this
surface, or the fragments were treated with 1-5 % (w/v) gelatin in water at 37 °C
and then the cells were cross linked to this surface with 2-5 % (v/v) glutaraldehyde in the cacodylate buffer for 60 min at room temperature. The cells
attached to the coverslip fragments were inserted into small envelopes made by
stapling together folded 'non-fluff' Velim tissue (General Paper and Box Co.,
from Koch-Light Laboratories Ltd., Colnbrook, Bucks., U.K.). The envelopes
reduced the loss of cells from the glass fragments during the following procedures. The samples were postfixed in 1 % (w/v) osmium tetroxide in the
cacodylate buffer for 60 min at room temperature, dehydrated in increasing
concentrations of either ethanol, or acetone. All were critical point dried in CO2
either direct form dehydration solutions or after transfer to amyl acetate in a
Polaron apparatus (Polaron Equipment Ltd., 60/62 Greenhill Crescent, Watford,
Hertfordshire, U.K.). After removal from the envelopes, the coverslips were
attached to the copper electron microscope carriers and good electrical contact
was established by painting around the fragment with conducting paste (Electrodag 915; Acheson Colloids Company, Prince Rock, Plymouth). The specimens
were coated with a 40-50 /tm thick layer of gold in a Polaron E 5000 Diode
sputtering system, and examined in a Jeol 100 CX electron microscope.
RESULTS
The results are presented in three sections. First, we describe the formation
of cell patterns in 4-cell-stage embryos. Second, we study the mechanisms which
modify these patterns during the formation of 8-cell-stage embryos. Third, we
280
C. F. GRAHAM AND E. LEHTONEN
20-48
20-46
20-57
16-45
18-00
20-56
19-15
19-32
19-53
00
16-45
22-30
100 pm
Fig. 1. For legend see opposite.
23-15
Cell patterns in mouse development
281
show how cell patterns at the 8-cell stage affect subsequent cell allocation to the
ICM and study cell surface characteristics during this process.
1. The formation of cell patterns at the 4-cell stage
We wished to find out if the form of the 4-cell embryo depended on cell
interactions which preceded that stage. The following results show that the
behaviour of the second cell to divide from the 2-cell stage (called the CD cell)
is directly related to the behaviour of the first cell to divide (called the AB cell).
We observed the division of the 4-cell stage in zona-free embryos to follow the
morphogenesis of the cell patterns; inside the zona pellucida, the cells were
closely packed and we could not see cell to cell contacts during the process of
division. For each embryo, we recorded the division movements of the AB cell
and then recorded the patterns of cells at the 4-cell stage between 60 and 90 min
after CD had divided. The patterns of the cells were described by the number of
cell contacts; the contact between two cells was scored as one contact. With the
intermittent 20 min observation intervals we did not see AB division behaviour
in between 20 and 40 % of the embryos in any experiment. The results were
similar for zona-free intact embryos and for embryos which had been dissociated
into single cells at the 2-cell stage and recombined 1-4 h before division; they
are described together here but recorded separately in Table 1.
Sometimes AB divided and pinched the membrane of the CD cell (Fig. 1 a);
frequently the membrane of the CD was pulled into the division groove as AB
divided but in a few cases a gap appeared between the daughters of AB (the A
and the B cell) and CD and then the CD membrane was pinched as A and B
flattened against each other. Either behaviour of AB was usually followed by the
formation of a 4-cell-stage pattern in which each cell was in contact with all
other cells of the embryo (a total of six cell-to-cell contacts; right-hand picture,
Fig. la).
Fig. 1. Formation of the 4-cell stage, (a) Formation of the six-total cell contact pattern.
As the AB cell divides so it pinches the cell membrane of CD. As CD divides so one
of the daughter cells moves on top and contacts all the cells of the embryo. (6) Formation of the five-total contact pattern. AB divides and appears to remain in contact
with CD. However, a gap appears between A and B and CD. As CD divides, one
of the daughter cells moves upwards but does not make contact with all the cells of
the embryo; it only touches one AB daughter, (c) Formation of a four-contact
pattern. AB divides so that one of its daughters initially does not make contact with
CD. Contact is re-established before CD divides and in this case only one daughter
of CD contacts the daughters of AB. {d) Formation of a different four-contact pattern.
AB divides and one daughter fails to contact CD before it divides. In this case the
daughters of CD both, make contact with both daughters of AB. Tracings from photographs. Dotted lines away from the observer (on the bottom of the dish); solid lines
either indicate that all cells are in the same plane or they indicate that cells are
towards the observer in embryos in which other cells have dotted outlines. Scale bar
is 100/*m. Time of photographs is indicated in hours, minutes.
282
C. F. GRAHAM AND E. LEHTONEN
Table 1. Formation of 4-cell-stage patterns
Number of cell-to-cell contacts in pattern
Six
Five
Four
15
(6)
1
(1)
-
2
(1)
22
(13)
2
(2)
1
7
(5)
7
(4)
Four
Three
Number of examples of
AB division pattern
29
6
(17)
(4)
The diagrams in the left-hand column illustrate the form of the division of AB (these are
illustrated in Fig. 1). The form of division of CD is illustrated in the other columns and the
data shows that the way in which AB divides is related to the way in which CD divides. The
AB cells are distinguished by a dot in the centre of the cell. The figures without parenthesis
refer to observations on all the embryos, while the figures within parenthesis indicate the
numbers of embryos which were dissociated and recombined before division.
Sometimes a gap appeared between A and B and CD; CD was not pinched
and this gap remained until CD divided (Fig. 1 b). When CD divided its daughters
(the C and the D cell) usually lay beside the daughter of AB in a rhomboid
pattern (a total of five cell-to-cell contacts; right-hand picture, Fig. \b). The
proportion of six to five total cell contact patterns which develop when AB
pinches CD is significantly different to the cases when there is a persistent gap
between A and B and CD (P < 0-001, ^ 2 on a 2 x 2 contingency Table with
Yates correction for small samples, Bailey, 1959).
AB could also divide in another way: it produced one daughter cell which
initially did not touch CD but before CD division this swung round and made
contact with CD (Fig. 1 c); in this case CD either divided so that only one of its
daughters touched an AB daughter (a total of four cell-to-cell contacts, righthand side, Fig. 1 c) or the rhomboid pattern was formed. The fourth possibility
was that AB divided and produced a daughter which never touched CD; in this
case CD frequently divided so that both C and D lay against only one daughter
of AB (another four total cell-to-cell contact pattern, right handdrawing, Fig. 1 d).
In the last two cases, where one AB daughter is initially formed away from CD,
the proportion of four and three to six and five cell-to-cell contact patterns is
Cell patterns in mouse development
283
Table 2. Four cell groups developing in intact embryos
and from 1/2 and 1/4 embryos
Number of four-cell groups with particular cell contact numbers
Treatment
Six
Five
Four
Three
Zona and embryos intact,
8-cell stage
Zona intact, one cell lysed at
the 2-cell stage
Zona off, embryos intact
Zona off, dissociated at the
2-cell stage
Zona off, dissociated at the
4-cell stage
_
15
4
1
8
2
-
-
4
5
9
8
10
5
5
4
11
28
14
5
The data records the total number of cell-to-cell contacts within a four-cell group which
developed from a single cell isolated at the 2-cell stage or 4-cell stage. The four-cell groups
developing from a single 2-cell-stage cell in the zona are more compact (have more six-contact
patterns) than the four-cell groups developing in the intact embryo.
significantly greater than in the first two cases where AB daughters do not show
this behaviour (P < 0-001, same test). Similarly the last two cases produce
significantly different types of four cell-to-cell contact patterns from each other
(P < 0-001, same test). Clearly there is an association between the form of AB
division and the form of CD division.
2. The formation of cell patterns at the 8-cell stage
At the 8-cell stage, the four descendants of each cell of the 2-cell-stage embryo
(a four-cell group) form patterns which can be described by the cell-to-cell
contacts within the group; this is the same procedure which was used to describe
the 4-cell-stage embryos in the previous section. The four-cell groups in the
intact zona contained 8-cell stage are natter than those at the 4-cell stage
(Graham & Deussen, 1978). We wished to find out if this flattening was due to
preceding cell interactions.
Single cells were isolated from 2-cell-stage embryos and cultured in large drops
to the 8-cell stage (Table 2). The four-cell groups formed from zona contained
isolated cells were more frequently in the six total cell-to-cell contact pattern;
there were significantly more of these patterns than were previously found in
intact 8-cell embryos (P < 0-001, same test; data of Graham & Deussen, 1978).
It therefore seemed likely that the two four-cell groups in each 8-cell-stage
embryo were held flat because they had stuck to each other during preceding
stages of development. This effect was not observed in the zona-free embryos.
There was no obvious change in the ability of isolated cells to form a range of
284
C. F. GRAHAM AND E. LEHTONEN
Table 3. Number of inside cells in 8- to 16-cell embryo
Number of cells in the embryo
Inside cells
8
9
10
11
12
13
14
15
16
0
1
2
15
0
0
2
0
0
2
0
0
2
0
0
2
1
0
0
1
0
0
1
0
1
1
2
0
4
10
An inside cell is defined as a cell which is not exposed to the medium and is completely
enclosed by the other cells. Previous estimates of the number of inside cells have used embryos
which have been cultured for long periods. The embryos in this table were all fixed within
30 min of the death of the mother. The numbers in the Table are the number of embryos in
the sample which contained either none or one or two inside cells. The results are from reconstructions of serially sectioned intact embryos.
15-45
1600
16-30
17-15
(b)
18-35
20-15
1845
50 Aim
Fig. 2. The movement of cells in four-cell groups in a monolayer. These are tracings
of photographs made on four-cell groups developing at an average temperature of
34 °C. This temperature slows down cell movement and cell division and makes it
easier to record cell position. The majority of records were made at 37 °C. Scale
bar is 50 fim. Time of photographs is indicated in hours, minutes.
Cell patterns in mouse development
285
Table 4. Formation of inside cells in four-cell groups
The cells which form an inside cell
Type of four-cell group
First
deep to
divide
Second
deep to
divide
First
superficial
to divide
Second
superficial
to divide
Intact four-cell group (n = 10)
8
1
1
—
Recombined four-cell group (n = 13)
10
5
2
The intact groups were rhomboid four-cell arrangements obtained from the 8-cell embryos
by partial dissociation. The recombined four-cell groups were obtained by completely dissociating the embryo to single cells and then recombining the cells in the rhomboid pattern.
Note that in the intact group only one inside cell was found while in the recombined group
an average of 1-3 inside cells were formed. An 'inside' cell in this situation is enclosed by
other cells but of course it is exposed to the tissue culture dish on one surface and the medium/
paraffin interface on the other. The definition of an inside cell here is different from that in
the intact embryo.
four-cell group patterns as development proceeded. Thus cells isolated at the
2- and 4-cell stage both form the patterns at a similar frequency in large drops
(Table 2).
3. Allocation of cells to the ICM
A. Formation of the ICM in vivo
Forty-two 8-to 16-cell-stage embryos were serially sectioned in order to estimate
the time of formation of inside cells in embryos developing in vivo (Table 3). In
these embryos inside cells were not formed until the 12-cell stage. The 16-cell
embryos usually had two inside cells.
B. Formation of inside cells in an experimental situation
To investigate the movements leading to cell allocation to the inside of the
embryo we studied the behaviour of groups of four 8-cell-stage cells in the fivecell-to-cell contact pattern; these were flattened in shallow drops so that the
movement of cells could be observed (see Materials and Methods). These fourcell groups were obtained either by dissociating the 8-cell embryo into two
groups of cells (intact four-cell group) or by dissociating the 8-cell embryo to
single cells and then recombining the cells in the five total cell-to-cell contact
pattern (recombined four-cell group). We did not know if these four cells were
derived from a single cell at the 2-cell stage. In most cases, the deep cells (with
three total cell contacts, Fig. 2, Table 4) formed the inside cells as these half
16-cell stages developed (24/27 cases), and the inside cells were usually formed
by the first of the deep cells to divide (18/24 cases).
19
EMB 49
286
C. F. GRAHAM AND E. LEHTONEN
Fig. 3. A four-cell group recombined from dissociated 8-cell-stage blastomeres.
After 5 h in culture the structure is well compacted. The distribution of microvilli
is relatively even.
Fig. 4. Higher magnification from the cell arrangement in Fig. 3. A zone with
relatively few microvilli aligns the cell contact areas. Short microvilli extend
frequently from one cell to another.
Fig. 5. An intact four-cell group after 4 h in culture. The arrangement was cultured
in a microdrop shallower than that in Fig. 3. (This was done in an attempt to mimic
the pressure conditions created by the other four blastomeres within the zona in vivo).
Fig. 6. Higher magnification from the four-cell group in Fig. 5. The density of
microvilli is greater towards the exterior surfaces of each cell. Microvilli extend
frequently from cell to cell.
Cell patterns in mouse development
287
Fig. 7. A recombined four-cell group cultured for 5 h in the same dish as the
arrangement in Fig. 3. The structure was fixed about 20min after division of the
other three-contact cell. The newly divided 16-cell stage cells appear less microvillous
than the 8-cell-stage blastomeres.
Fig. 8. A higher magnification from the arrangement in Fig. 7. The upper 16-cell
stage cell has maintained all three cell contacts of its mother cell, the lower one
is in contact with an 8-cell-stage blastomere through microvilli.
Fig. 9. A recombined four-cell groupfixedabout 1 h 30 min after the division of the
other two-contact cell. The density of microvilli on the surfaces of the 16-cell
blastomeres appears to be slightly less than that of the 8-cell stage blastomeres.
Fig. 10. A higher magnification from the arrangement in Fig. 9. The density of microvilli on the 16-cell blastomeres (the two lower cells) seems to be greater than that on
the 16-cell blastomeres of Fig. 8.
288
C. F. GRAHAM AND E. LEHTONEN
C. Cell surface characteristics at the 8- to 16-cell stage
In order to explain the mechanism by which cells are allocated to the ICM we
studied cellular relationships in 22 8-cell embryos, 13 8- to 16-cell embryos and
five later morulae with a SEM. Also cell arrangements similar to those described
in paragraph 3B were studied at different stages of their development.
Our observations on the surface structure of intact, zona-free embryos
accorded with the earlier reports (Calarco & Epstein, 1973; Ducibella, Ukena,
Karnovsky & Anderson, 1977). The blastomeres showed microvillous projections, and the density of microvilli and their distribution varied to some
extent. Microvilli were regularly seen to extend over the eel) borders and to reach
the surface of the neighbouring cell, but long microvilli (3-4 /«n long) were very
rare, although one or two were present in most embryos.
We cultured four-cell groups in the rhomboid pattern and fixed them before
and after division of some cells in the group. Six four-cell groups were examined
before division (Figs. 3-6); in general their surface characteristics were similar
to those of intact embryos. Before compaction, the distribution of microvilli on
the cell surface was usually even but after compaction a relatively smooth zone
lay on either side of the line of cell contact (Figs. 3-4). In one case the microvilli
were concentrated on the exterior surface of each cell in the group (Figs 5-6);
this distribution is probably an artifact because this group was cultured in a
particularly shallow microdrop.
Thirteen four-cell groups were examined after at least one cell in the group
had divided. In two cases only one deep cell had divided: the interior daughter
maintained the three cell contacts of the parental cell and the exterior daughter
lay beside another cell in the group (Fig. 7). This group was fixed 20 min
after division and the recently divided cells appeared to have fewer and shorter
microvilli than the undivided cells in the group (Figs. 7-8). In two cases only
one superficial cell had divided: either one (Fig. 11) or both (Fig. 9) of its
daughters remained in contact with the other cells of the group. Close packing
of the cells was obvious. The newly divided cells appeared to have slightly
fewer microvilli than the other cells in the group when fixed 90 min after division
(Fig. 10) but this difference was not obvious at 150 min after division (Fig. 11).
In the thirteen four-cell groups which had freshly divided cells we never observed
the segregation of microvilli to the exterior daughter of a recently divided cell.
DISCUSSION
We argue that the behaviour of cells during early cleavage principally depends
on their interactions with other cells in the embryo during the preceding interphase.
Cell patterns in mouse development
289
Fig. 11. A detail from a cell arrangement fixed about 2 h 30 min after cell divisions.
The structure is compacted, the distribution of microvilli is relatively even. The
density of microvilli on the surface of an 8-cell blastomere (right lower cell) is comparable to that of the three 16-cell blastomeres.
1. The 2- to 4-celI division
The correlation between the form of the AB division and the form of the CD
division suggests that there is a causal relationship between these two events.
However, it could be argued that this relationship was due to the enzyme used
to remove the zona pellucida. It was noticed that the loose four and three cellto-cell contact patterns were frequently observed among embryos exposed to
pronase for long periods until the zona was completely dissolved. It might be
that the correlation was due to a similar degree of degradation of surface
molecules on both cells of a particular embryo. For several reasons we consider
this an unlikely explanation of our results. First, six and five cell-to-cell contact
patterns are observed in zona contained intact embryos (e.g. Lewis & Wright,
1935; Graham & Deussen, 1978). These have never been exposed to pronase and
therefore the distinction between these two does not depend on a deliberately
applied proteolytic enzyme. Second, in most experiments at least one embryo
formed each of the six, five, or four cell-to-cell contact patterns and all embryos
were exposed to pronase for an identical time.
It might also be argued that the orientation of the CD daughters was initially
19-2
290
C. F. GRAHAM AND E. LEHTONEN
random and that they vibrated relative to A and B; since the orientation of the
AB daughters varied with respect to CD before its division, they would provide
different sizes of collision targets and generate the cell patterns. Apart from our
failure to observe any vibration of the CD daughters, this mechanism would not
account for the generation of the six and the five cell-to-cell contact patterns
where the A and the B target is identical with respect to CD (top two lines in
Table 1 and Fig. 1 a, b).
We interpret our results as evidence of a causal relationship between the form
of the AB division and the form of the CD division. The similarity of the results
with intact embryos and with embryos which have been dissociated and reaggregated, suggests that the molecular mechanisms which relate the forms of
AB and CD division depend on continous cell interactions. These interactions
need only occur in the 30 min to 4 h time interval between reaggregation and
cell division.
It is probable that only brief contacts of A and B with CD determine the
pattern of cells at the 4-cell stage. If AB divides so that only one daughter touches
CD, then the forms in the two right-hand columns of Table 1 are produced.
However, if AB divides so that initially only one daughter touches CD and then
both daughters touch CD (Fig. 1 c) at the last observation interval before CD
division, then the forms on the left three columns of Table 1 are produced. It
seems that brief cell interactions generate the forms of the 4-cell-stage embryos
which have been observed.
2. The 4- to 8-cell stage
Inside the zona pellucida, the four descendants of each cell of the 2-cell-stage
embryos usually lie flat in the five cell-to-cell contact pattern at the 8-cell stage
(Graham & Deussen, 1978). However, when one cell is lysed inside the zona,
then the majority develop the six cell-to-cell contact pattern (Table 2). We
conclude that the flattening of each four-cell group in the intact embryo is not
a consequence of any inherent change in the capacity of cells to form particular
patterns. It is more likely that the flattening of each four-cell group was due to
the fact that it was stuck to the other four-cell group in the intact embryo. This
interpretarion is favoured by the observations on zona-free embryos. In these,
there were not significantly more six total cell-contact patterns in four-cell
groups developing from isolated 2-cell-stage cells when compared to intact
embryos. This is probably because loose patterns develop in 4-cell-stage zonafree embryos and thus the four-cell groups are not held flat against each other
during development to the 8-cell stage. These experiments demonstrate that the
cell contacts before the 8-cell stage influence cell patterns at the 8-cell stage.
Cell patterns in mouse development
291
3. The mechanism of inner cell formation
In intact embryos which have been freshly isolated or which have been cultured
for long periods, inside cell formation begins at the 8- to 16-cell stage (Table 3;
Mulnard, 1967; Barlow, Owen & Graham, 1972) and continues during later
stages of development (Graham & Deussen, 1978). The allocation of cells to the
inside of the embryo is related to their division order and relative position
(Kelly et al. 1978; Graham & Deussen, 1978). These features of the normal cell
lineage are reproduced by the behaviour of the four-cell groups. Unlike the
intact embryo, the relative positions of cells in recombined four-cell groups does
not depend on their prehistory. Therefore the regularities of inside cell formation
in this experimental situation (Table 4) result from interactions between the cells
in the new arrangement.
We have to explain two features of the cell lineage and the behaviour of fourcell groups: (a) deep cells at the 8-cell stage contribute disproportionately more
cells to the inside than do superficial cells; (b) first dividing deep cells contribute
inside cells more frequently than last dividing deep cells.
The explanation for (a) appears to be that daughters of deep cells form inside
cells because they are formed near the interior of the embryo or group. However,
starting from an analogous cell arrangement, a single macromere moves to and
is retained in the inside of the mollusc Patella vulgata (van den Biggelarr, 1977),
and in this case continuous cell interactions probably account for this behaviour
(van den Biggelarr & Guerrier, 1979). The explanation for (b) requires a consideration of the cellular events in the four-cell group. The first deep cell to divide
elongates radially and divides so that the daughter cells are distributed radially
(Figs. 2, 7). The division plane did not appear to divide this cell across its
longest axis in the preceding interphase (contrary to a strict interpretation of
Hertwig's (1885) rule); rather it appears that this deep cell maintains cell contacts
through division and can only elongate away from other cells. In this case
the division plane would be imposed on the dividing cell by its previous cell
contacts (compare Rappaport, 1974). We think, but cannot prove, that similar
constraints determine the distribution of the daughter of the other cells in the
group. Sometimes inside cell formation is completed soon after each cell in the
group has divided (Fig. 2a). Usually inside cell formation occurs long after
division (Fig. 2b) and in both situations we have to explain how the other cells
in the embryo are drawn around the first deep cell to divide and enclose its
interior daughter.
We next attempt to discriminate between various hypotheses which could
account for this second movement using the SEM observations. First, it is
possible that the first deep cell to divide and its daughters retain tenuous microvillar contact with all other cells in the group; then microvilli might contract
and draw the other cells around the deep dividing cell (Fig. 12a). Such microvilli
might not be seen by light microscopy. However, we exclude this mechanism
19-3
292
C. F. GRAHAM AND E. LEHTONEN
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Fig. 12. Models of inside cell formation, (a) The first model depends on long contractile microvilli. As the first deep cell to divide elongates radially (1), so long microvilli are stretched and contract subsequently to catapult Ihe interior daughter to the
inside of the group (3 and 4). The inwards moment of the contraction of these microvilli will be greater than that of microvilli acting on the opposite cell because of the
angles of the two sets of microvilli. (b). The second model depends on the segregation of microvilli to the outside daughter of the first deep cell to divide. All cells
start with an equal density of microvilli (1). The first deep cell to divide is cleaved
tangentially and its exterior daughter inherits a greater density of microvilli than all
the other cells on the group which divide radially. If the pull exerted by the cells
on each other depends on microvillar density then the exterior daughter will draw
the other cells of the group around it (closing the purse strings), (c) The third model
also depends on unequal densities of microvilli. It is assumed that microvillar
density increases with time during a particular cell stage. The top and right cell in
the group are depicted with twice the number of microvilli when compared to
the other two cells in the group. They divide first and draw the other cells in the
group around them if it is assumed that the strength of pull between cells depends
on microvillar density.
both because very long microvilli (10 /on) have only once been seen (Calarco &
Epstein, 1973) and were not observed in the intact embryos in this or previous
studies (Ducibella et al. 1977), and because such processes were not found in the
cell groups (Figs. 3-8).
Second, it is possible that as the first deep cell divides, so microvilli segregate
to the exterior daughter cell. If the force with which cells can pull on each other
is related to microvillar density, then this cell would pull the other cells of the
embryo around it (Fig. 126). The suggestion that microvilli may pull cells
together in the embryo has been made previously (Ducibella et al. 1977) and this
paper also showed that microviJJi are concentrated on the exterior surface of
Cell patterns in mouse development
293
8-cell-stage cells. This mechanism might operate in the intact embryo but it
cannot explain the behaviour of the four-cell groups. Microvilli did not segregate to the outside daughters of cells which divide at right angles to the radius
of the group (Figs. 8 and 11) and we never observed an uneven distribution of
microvilli on the individual cells of each of the 13 divided four-cell groups which
were studied.
We consider that cell cycle dependent changes in cell membranes are likely
to be the events which are involved in the observed cell behaviour. The behaviour
of the cells may depend on the density of microvilli. In the mouse embryo (this
study; Ducibella & Anderson, 1975; Ducibella et ah 1911), and in somatic cells
in culture (Porter, Prescott & Frye, 1973; Knutton, Summer & Pasternak, 1975),
cells early in the cell cycle have fewer microvilli than cells late in the cell cycle.
We again assume that the cells pull on each other with contractile microvilli and
the more microvilli the greater the pull. During the 16-cell stage, the cells which
have been longest at that stage will then have the greatest pull and tug the cells
around them (Fig. 12 c). There is another cell cycle dependent event which might
account for the cell behaviour either by itself of in combination with the previous
mechanism. Blastomeres round up preceding division and subsequently flatten
against each other after division. The contact area between blastomeres increases
during the cell cycle (Lehtonen, unpublished results), and so during the 16-cell
stage the cells longest at that stage will have the greatest area of contact with
other cells. If either mechanism were to operate earlier in development then it
would explain the interior position of the first cells to reach the 8-cell stage
(Graham & Deussen, 1978) and if it were to operate later it would explain why
the daughters of fast dividers make a disproportionately large contribution to
the ICM after reaggregation at the 8-cell stage (Kelly et al. 1978).
CONCLUSIONS
1. Cell patterns at the 4-cel.l stage are related to the division movements of
the first cell to divide from the 2-cell stage. This cell needs only to be in contact
with the other cell during a short period for this relationship to occur.
2. Cell patterns at the 8-cell stage depend on cell interactions preceding that
stage.
3. Cell patterns at the 16-cell stage are related to cell contacts at the 8-cell
stage. This relationship also occurs when the cell contacts are established a short
time before division to the 16-cell stage.
4. Scanning electron microscope observations suggest that cell cycle-dependent alterations in microvillar density may account for the allocation of cells
to the inner cell mass and trophectoderm. The increase in contact area between
cells during the cell cycle may also be involved in this event.
We would like to thank the MRC for funding these studies, EMBO for providing a
Fellowship for E.L., and Marie Dziadik, Virginia Papaioannou, and Richard Gardner for
294
C. F. GRAHAM AND E. LEHTONEN
checking the manuscript. We were particularly grateful to Ms Margaret K. Arnold and
Dr K. A. Harrap of the Natural Environment Unit of Invertebrate Virology, Oxford, for the
use of the Jeol 100 CX Temscan electron microscope.
REFERENCES
BAILEY, N. J. J. (1959). Statistical Methods in Biology. London: English Universities Press.
BARLOW, P. W., OWEN, D. A. S. & GRAHAM, C. F. (1972). DNA synthesis in the preimplantation mouse embryo. /. Embryol. exp. Morph. 27, 431-445.
BIGGELARR VAN DEN, j . A. M. (1977). Development of dorsoventral polarity and mesentoblast determination in Patella vulgata. J. Morph. 154, 157-186.
BIGGELARR VAN DEN, J. A. M. & GUERRIER, P. (1979). Dorsoventral polarity and mesentoblast determination as concomitant results of cellular interactions in the mollusc Patella
vulgata. Devi Biol. (In the Press.)
CALARCO, P. G. & EPSTEIN, C. J. (1973). Cell surface changes during preimplantation
development in the mouse. Devi Biol. 32, 208-213.
DUCIBELLA, T. & ANDERSON. E. (1975). Cell shape and membrane changes in eight cell mouse
embryo: prerequisite for morphogenesis of the blastocyst. Devi Biol. 47, 45-58.
DUCIBELLA, T., UKENA. T., KARNOVSKY, M. & ANDERSON, E. (1977). Changes in cell surface
and cortical cytoplasmic organization during early embryogenesis in the preimplantation
mouse embryo. J. Cell Biol. 74, 153-167.
GRAHAM, C. F. & DEUSSEN, Z. A. (1978). Features of cell lineage in preimplantation mouse
development. /. Embryol. exp. Morph. 48 (in press).
HERTWJG, O. (1885). Welchen Einfluss iibt die Schwerkraft auf die Theilung der Zellen? Jena.
Z. Natur. 18, 175-205.
KELLY, S. J., MULNARD, J. G. & GRAHAM, C. F. (1978). Cell division and cell allocation in
early mouse development. J. Embryol. exp. Morph. 48 (in press).
KNUTTON, S., SUMNER, M. C. B. & PASTERNAK, C. A. (1975). Role of microvilli in surface
changes of synchronized P8157 mastocytoma cells. /. Cell Biol. 66, 568-576.
LEWIS, W. H. & WRIGHT, E. S. (1935). On the early development of the mouse egg. Contr.
Embryol. Carnegie Inst. 148, 115-143.
MCKEEHAN, W. L. & HAM, R. G. (1976). Stimulation of clonal growth of normal fibroblasts
with substrata coated with basic polymers. J. Cell Biol. 71, 727-734.
MINTZ, B. (1967). Mammalian embryo culture. In Methods in Development Biology (ed.
F. H. Wilt & N. K. Wessels) pp. 379-400. New York: T. Y. Crowell.
MULNARD, J. G. (1967). Analyse rnicrocinematographique du developpement de l'oeuf de
souris du stade II au blastocyste. Archs Biol. {Liege) 78, 107-138.
PORTER, K., PRESCOTT, D., & FRYE, J. (1973). Changes in surface morphology of Chinese
hamster ovary cells during the cell cycle. /. Cell Biol. 57, 815-836.
RAPPAPORT, R. (1974). Cleavage. In Concepts of Development (ed J. Lash & J. R. Whittaker),
pp. 76-98. Stamford: Sinauer Associates.
TARKOWSKI, A. K. & WROBLEWSKA, J. (1967). Development of blastomeres of mouse eggs
isolated at the 4- and 8-cell stage. /. Embryol. exp. Morph. 18, 155-180.
WHITTEN, W. K. (1971). Nutrient requirements for culture of preimplantation embryos in
vitro, (ed. G. Raspe). Advances in the Biosciences 6, 129-140.
WILSON, I. B., BOLTON, E. & CUTTLER, R. H. (1972). Premplantation differentiation in the
mouse egg as revealed by microinjection of vital markers. /. Embryol. exp. Morph. 27,
467-479.
(Received 9 August 1978, revised 28 September 1978)