Development 100, 325-332 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
325
When and how does cell division order influence cell allocation to the
inner cell mass of the mouse blastocyst?
C. LOUISE GARBUTT, MARTIN H. JOHNSON and MARTIN A. GEORGE
Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK
Summary
Aggregate 8-celI embryos were constructed from four
2/8 pairs of blastomeres, one of which was marked
with a short-term cell lineage marker and was also
either 4 h older (derived from an early-dividing 4-cell)
or 4h younger (derived from a late-dividing 4-cell)
than the other three pairs. The aggregate embryos
were cultured to the 16-cell stage, at which time a
second marker was used to label the outside cell
population. The embryos were then disaggregated
and each cell was examined to determine its labelling
pattern. From this analysis, we calculated the relative
contributions to the inside cell population of the
16-cell embryo of older and younger cells. Older cells
were found to contribute preferentially. However, if
the construction of the aggregate 8-cell embryo was
delayed until each of the contributing 2/8 cell pairs
had undergone intercellular flattening and then had
been exposed to medium low in calcium to reverse this
flattening immediately prior to aggregation, the advantage possessed by the older cells was lost. These
results support the suggestion that older cells derived
from early-dividing 4-cell blastomeres contribute
preferentially to the inner cell mass as a result of
being early-flattening cells.
Introduction
division is called differentiative, and yields an outer
polar cell derived from the apical region of the parent
cell and an inner apolar cell derived from the basolateral region (Johnson, 1986). Not all polar 8- or 16-cell
blastomeres divide differentiatively, some cleaving
along, rather than across, the axis of polarity so
yielding two similar progeny both of which are polar
and outside. Such a division is called conservative. The
number of inside cells within an individual embryo
will therefore be determined by the ratio of differentiative to conservative divisions by polarized cells at the
8- to 16-cell and 16- to 32-cell transitions. It has been
shown in the intact embryo that once cells are
deposited internally as a result of a differentiative
division, they rarely if ever re-emerge to assume an
outside position and fate (Pedersen et al. 1986;
Fleming, 1987; Dyce et al. 1987).
The allocation of cells to the ICM lineage during
cleavage is influenced by the division order of the
cells, the progeny of early-dividing cells making a
preferential contribution (Kelly, Mulnard & Graham,
1978; Surani & Barton, 1984). Such a preferential
contribution could be achieved if early-dividing 1/8
The mouse expanded blastocyst contains two distinct
and committed cell subpopulations, the trophectoderm and the inner cell mass (ICM). The allocation of
cells to the ICM and trophectoderm lineages depends
upon their internal or external position within the
embryo at earlier stages (Tarkowski & Wroblewska,
1967; Hillman, Sherman & Graham, 1972). It has
been known for some time that the earliest stage at
which inside cells can be detected is the 16-cell morula
(Barlow, Owen & Graham, 1972) and recent evidence suggests that in the undisturbed embryo this
pool of inside cells contributes on average 75 % of the
ICM cells. The remaining 25 % of ICM cells derives
almost exclusively from a second allocation of cells to
the inside in the 32-cell embryo (Balakier & Pedersen, 1982; Pedersen, Wu & Balakier, 1986; Fleming,
1987; Dyce, George, Goodall & Fleming, 1987). The
allocation of cells to an inside position is achieved by
the division of an asymmetrically organized (polarized) 8- or 16-cell blastomere such that the progeny
do not receive equivalent endowments. Such a
Key words: mouse, compaction, division order, lineage,
ICM, cell division.
326
C. L. Garbutt, M. H. Johnson and M. A. George
or 1/16 polar cells tended to divide differentiatively
(yielding one inside and one outside cell) rather than
conservatively (yielding two outside cells). Since it is
now known that an average 75 % of ICM cells is
derived from the inside cell population formed at the
8- to 16-cell transition, we have examined whether
those 1/8 blastomeres that are formed first within the
embryo might divide differentiatively to yield inside
cells more frequently than do later-forming 1/8
blastomeres. We have found (1) that earlier-formed
blastomeres do indeed contribute cells preferentially
to the inside cell population at the 16-cell stage and
(2) that this preferential contribution arises because
the cells that are formed early undergo intercellular
flattening in advance of the other cells.
Materials and methods
(A) Embryo collection and culture
Female MF1 mice (Central Animal Services, University of
Cambridge) were superovulated by an injection of 5i.u.
pregnant mares' serum (PMS) followed 45-48 h later by
5i.u. human chorionic gonadotrophin (hCG). The females
were paired overnight with HC-CFLP males (Hacking and
Churchill Ltd). The presence of a vaginal plug the next
morning indicated that a mating had occurred. Embryos
were collected at the 2- to 4-cell stage by flushing oviducts
52 h post-hCG with prewarmed (37°C) medium 2 containing 4 m g m F ' bovine serum albumin (M2+BSA; Fulton &
Whittingham, 1978). Zona-intact embryos were cultured in
pre-equilibrated medium 16 containing 4mgml~1 bovine
serum albumin (M16+BSA; Whittingham, 1971) in Falcon
culture dishes, under oil at 37°C and 5 % CO2 in air. Sterilin
culture dishes were used for culture of zona-free embryos
and single cells.
(B) Embryo disaggregation and reaggregation
Removal of the zona pellucida was achieved by exposure of
embryos to prewarmed acid Tyrode's solution for about 20 s
(Nicolson, Yanagimachi & Yanagimachi, 1975). Embryos
at the 4-cell stage were disaggregated byfirstdecompacting
them by incubation in prewarmed (37°C) Ca2+-free M2
plus 6mgml~ 1 BSA for 10-15 min and then pipetting them
gently through a flame-polished micropipette. To disaggregate 16-cell embryos, the Ca2+-free M2+BSA was supplemented with a 1 in 10 dilution of 5 % (w/v) trypsin and
2% (w/v) EDTA (Gibco Ltd; Fleming, 1987).
Individual 4-cell blastomeres (1/4 cells) were cultured
and examined every hour for evidence of division to 2/8
pairs. Any such pairs were harvested and stored for 0-11 h
until they were used to make aggregate embryos. Four 2/8
pairs were decompacted by a 5 min incubation in Ca2+-free
M2+BSA then exposed briefly to phytohaemagglutinin
(PHA; Gibco Ltd) diluted 1:20 in M2+BSA to increase
their adhesiveness. Aggregation was carried out as described in Kelly et al. (1978). First, the 2/8 pairs were
arranged in two sets of four cells with each pair lying
parallel to its neighbour so as to form a 'square' quartet.
When these sets had adhered, one set was placed on top of
the other but rotated through 45° so that all the eight cells in
each aggregate embryo had similar positions relative to
each other. Most aggregate embryos were cultured until
the mid-16-cell stage before surface labelling and disaggregation. Some aggregate embryos were fixed during the
8-cell stage for histological analysis.
(C) Labelling procedures
A stock solution of carboxylated fluorescent latex microparticles (yellow-green latex, 2-5 % solids, 0-06 fim particle
diameter; Fluoresbrite, Polysciences) was diluted 1:20 in
M2+BSA. Intracellular labelling of endocytic organelles
was achieved by incubating 2/8 cells in the label for 20 min,
then washing in M2+BSA and culturing for 2h. Preliminary experiments confirmed that labelling with latex did not
affect development (Fleming & George, 1986).
Surface labelling of exposed cells in 16-cell aggregate
embryos was achieved by exposure to concanavalin A
conjugated with tetramethylrhodamine isothiocyanate
(TRITC-ConA, Polysciences) at lmgrnP' M2+BSA
for lmin (Fleming & George, 1986). To prevent sticking
during labelling, embryos were kept clear of the bottom of
the culture dish.
(D) Microscopy
Cells were examined only from those embryos where all 16
blastomeres had been recovered successfully. Thus embryos were discarded if they either contained arrested 1/8
cells or suffered some cell loss or lysis during disaggregation. Cells were fixed in 4 % formaldehyde in phosphatebuffered saline (PBS) for 20 min and stored in M2+BSA at
4°C. The fixed cells were transferred to drops of M2+BSA
under oil in wells of a tissue-typing slide (Baird and
Tatlock) and overlain with a cover slip.
Some aggregate 8-cell embryos were fixed, embedded in
JB4 water-soluble resin and sectioned as described in
Fleming & George (1986). Sections were viewed by differential interference and fluorescence microscopy.
Material was viewed on a Leitz Ortholux II microscope
with filter sets N2 for rhodamine and L2 for FITC.
Photomicrographs were taken on Kodak Tri-X film using a
Leitz Vario-orthomat photographic system.
Results
(A) Older 8-cells contribute preferentially to the inside
cell population at the 16-cell stage
Pairs of newly formed 2/8 blastomeres were recovered at hourly intervals from a pool of isolated 1/4
blastomeres. Some of these were labelled with green
fluorescent latex, whilst other pairs remained unlabelled. This procedure was continued for up to 7h.
Aggregates were made of one labelled pair of cells
with three unlabelled pairs of cells. In some aggregates the labelled pair was 4h postdivision and the
unlabelled pairs were newly divided (labelled cells
designated older), whilst in others the labelled pair
was newly divided and the unlabelled pairs were 4h
postdivision (labelled cells designated younger). The
Division order and cell allocation
aggregate embryos were then cultured to the mid- to
late-16-cell stage, at which point the embryos were
incubated briefly in rhodamine-conjugated concanavalin A to label the exposed outside cells. The
embryos were then disaggregated to groups of between one and four cells. The cells were examined for
their red and green fluorescent staining patterns, as a
result of which each cell was assigned to older or
younger groups and to inside or outside positions
(Fig. 1). From this information, it was possible to
deduce (1) the ratio of inside to outside cells in each
aggregate, and (2) the proportion of labelled 1/8
blastomeres that had divided differentiatively.
327
In both types of aggregate, the number of inside
cells varied between 2 and 7 with mean values of 5-0
and 4-3 inside cells present in aggregates containing
labelled older and younger cells, respectively. These
figures are comparable with those reported previously for intact MFl-strain embryos (range 2 to 7;
mean 5-2, Fleming, 1987), suggesting that the aggregate embryos approximate to normal developmental
behaviour. The distribution of inside cell numbers in
the two groups of aggregates did not differ significantly (Mann-Whitney U-test, P > 0 - 0 5 ) . Since the
two populations of embryos were comparable with
respect to their patterns of inside: outside cell ratios,
they were next compared for the relative allocation
t.
Fig. 1. (A,B) Nine 16-cell blastomeres isolated from an aggregate embryo made by aggregating four 2/8 pairs together
and culturing the embryo to the mid- to late-16-cell stage. One pair of 2/8 blastomeres (an early-formed pair) was
labelled with fluoresceinated latex and the four descendant labelled cells are among these nine, as shown in E,F. Prior
to disaggregation the intact 16-cell embryo was incubated briefly in rhodamine-labelled concanavalin A to mark the
outside cells; six of the cells shown are labelled as shown in C,D. Cell number 1 is unlabelled by either marker and is
thus an inside cell derived from a later-formed 2/8 pair. Cells numbered 2 are double labelled and are thus outside cells
derived from the early-forming 2/8 pair. Cells numbered 3 are labelled only with rhodamine and are thus outside cells
derived from a later-formed 2/8 pair. Cells numbered 4 are labelled only by fluorescein and are thus inside cells derived
from the early-forming 2/8 pair. X400.
328
C. L. Garbutt, M. H. Johnson and M. A. George
Table 1. Influence of the division order and time of aggregation on the incidence of differentiative and
conservative divisions by mouse 8-cell blastomeres
Aggregation protocol
(1) Immediate aggregation
Labelled
cells
No. of labelled
1/8 cells
analysed
•Number of
conservative
divisions (%)
'Number of
differentiative
divisions (%)
Older
Younger
44
56
11(25)
36(64)
33 (75)
20(36)
X2 test: significantly different P < 0-001
(2) Delayed aggregation
Older
Younger
28
24
14(50)
16 (67)
14(50)
8(33)
X2 test: not significantly different P > 0 0 5
* A conservative division of a 1/8 blastomere yields two outer polar cells, whilst a differentiative division yields one outer polar and
one inner apolar cell.
of labelled cells to the inside population (Table 1,
line 1). It is evident that the older cells contributed
relatively more cells to the inside than did younger
cells as a result of a significantly greater proportion of
differentiative divisions.
(B) The advantage possessed by older cells is related
to their intercellular flattening properties
Observation of aggregated embryos for the first few
hours of their period in culture revealed that the
cluster of older cells flattened in advance of the
cluster of younger cells (Fig. 2A,B)- We examined
whether this early flattening might be responsible for
the preferential allocation of cells to the inside cell
population.
Pairs of newly formed 2/8 blastomeres were harvested and some were labelled, exactly as described
above. However, the aggregate embryos were not
constructed immediately. Rather, the 2/8 pairs were
cultured individually for either 7h (younger) or 11 h
(older) by which time intercellular flattening had
occurred between the two component cells of each
pair, regardless of their relative age. One labelled
(older or younger) and three unlabelled (younger or
older respectively) 2/8 pairs were then decompacted
by brief exposure to medium low in calcium and then
aggregated together as above. The aggregates were
placed in culture, where all cells were observed to
flatten on each other simultaneously. The compacted
aggregate embryos were cultured to the mid- to late16-cell stage and then analysed as described above.
The distributions of inside: outside cell ratios in
embryos with labelled older and younger cells did not
differ significantly (labelled older aggregates: range 2
to 6, mean 4-0; labelled younger aggregates: range 3
to 6, mean 4 1 ; not significantly different by MannWhitney U-test, P > 0 0 5 ) . The two groups of embryos were therefore compared for the relative allocation of labelled cells to the inside population
(Table 1, line 2). The difference between the two
types of embryo was not significant.
(C) Histological examination of aggregate embryos
Aggregate embryos were made in which a labelled
2/8 pair was combined with three unlabelled pairs in
reciprocal combinations as described in section Al.
Embryos were harvested 7h after aggregation, by
which time the 8-cell aggregates were fully compacted. Embryos were fixed and sectioned serially,
and the relationship between the labelled and unlabelled cells was determined. Fig. 2C-F shows representative patterns. In all five aggregates examined
in which the labelled cells were older, labelled cells
were found deep within the compacted embryo
(Fig. 2C,D), whereas this pattern was observed for
only two labelled cells in seven aggregates in which
the labelled cells were younger, all other labelled cells
being located superficially (Fig. 2E,F).
Discussion
The principle underlying these experiments is very
simple. If cells enter a particular developmental cell
cycle at different times and if cells acquire a particular
property at a certain point in that cell cycle, then
the early-formed cells will acquire that property in
advance of cells formed later. Temporal heterogeneity will lead to physical heterogeneity. If the cellcycle-linked property affects cell shape or interaction,
the physical differences could also generate spatial
heterogeneity. Thus, differences in cell division order
could generate spatial pattern within the embryo.
Such a mechanism may operate to influence the
establishment of the embryonic: abembryonic axis of
the mouse embryo (Garbutt, Chisholm & Johnson,
1987). Here we examine whether and how the same
Division order and cell allocation
Fig. 2. (A,B) An aggregate embryo made up of three
early-formed 2/8 pairs aggregated to one later-forming
2/8 pair that had been labelled with FITC-latex. The
aggregate had been cultured for 4h. Note that the earlyformed unlabelled cells that are now 8 h into the cell
cycle areflatteningon each other and that the two
labelled later-forming cells are not yet showing evidence
of intercellularflattening.(C,D) Sections through
aggregate embryos 7 h after they were constructed by
aggregating a 4h old, labelled 2/8 pair with three Oh old,
unlabelled 2/8 pairs. Note that one labelled cell is located
deep in the embryo and with a radially elongated shape.
(E,F) A section through an embryo constructed in a
reciprocal manner. In this embryo the labelled cells are
superficial and tangentially attenuated, a characteristic of
most late-dividing cells. x400.
principle might operate to set up the earlier radial
(inside: outside) axis in the mouse embryo.
Previous experiments have suggested that such a
mechanism might be operating to influence cell allocation in the mouse blastocyst, early-dividing cells
contributing a disproportionate number of progeny to
the ICM (Kelly et al. 1978; Surani & Barton, 1984).
329
Since the allocation of cells to an inside position and
thus to the ICM lineage is achieved by the differentiative division of outer polar 1/8 and 1/16 blastomeres, and since it is dividing 1/8 cells that provide
the majority of the inside cells that go to form the
ICM, we examined whether those 1/8 cells that
formed first cleaved differentiatively more frequently. We aggregated four pairs of 8-cell blastomeres differing in age by 4h, a difference within the
age range of cells in nonmanipulated embryos (Kelly
etal. 1978; Smith & Johnson, 1986). Our results show
that 16-cell embryos derived from such aggregates
have a similar range and mean number of inside cells
to nonmanipulated embryos (Fleming, 1987). The
results also support the suggestion that 8-cell blastomeres only contribute cells to the inside by a process
of differentiative division, since the labelled pair of
2/8 cells was observed to contribute a maximum of
two and a minimum of zero inner cells. Our results
also reveal a clear advantage of early-formed cells
over cells formed later in their relative contribution to
the inside cell lineage.
The inside cells that are generated at the 8- to 16cell transition give rise to about 75 % of the ICM cells
on average (Fleming, 1987). It seems likely that the
second smaller allocation of cells to the embryo
interior that can occur at the subsequent cell division
shows a similar preferential contribution from the
early-dividing cells, since a comparison between our
results on the proportion of the inside cells at the 16cell stage generated from early-dividing cells with the
results reported by Kelly et al. (1978) for the equivalent proportion of cells in the ICM of the blastocyst
reveals that they do not differ significantly (MannWhitney U-test, P>0-05). We must ask what property of early-dividing cells results in their divisions
being differentiative, to yield one inside and one
outside cell, rather than conservative, to yield two
outside cells.
This question was considered by Graham and his
colleagues, who showed that early-dividing cells
tended to establish more contacts with other cells and
to lie deeper within the embryo (Graham & Deussen,
1978; Graham & Lehtonen, 1979). This observation
was confirmed in the histological study reported here.
Graham and his colleagues therefore suggested that
some property associated with cell surface adhesion
might be important in determining the preferential
internal location of early-dividing cells and their
progeny. Indeed, it has been demonstrated that
intercellular adhesive properties do change over a
defined period during the fourth cell cycle such that
after about 7h into the cycle all cells are flattened
maximally upon each other (Lehtonen, 1980; Ziomek
& Johnson, 1980). The asynchrony of flattening in
aggregates of early- and late-dividing cells observed
330
C. L. Garbutt, M. H. Johnson and M. A. George
here (Fig. 2A,B) confirms that this cell cycle dependence of the flattening process is indeed cell autonomous and related to the time of entry into the fourth
cell cycle. However, a number of other cellular and
intercellular features also develop at defined periods
during the fourth cell cycle, notably the appearance
of functional gap junctions (Goodall & Johnson,
1982, 1984) and the polarization of the cytoskeleton,
cytoplasmic organelles and surface of individual
blastomeres (reviewed in Johnson & Maro, 1986).
Moreover, the changes in cell adhesion that occur
during the fourth cell cycle influence the changes in
junctional communication and polarization (Goodall,
1986; Johnson, Maro & Takeichi, 1986). The problem
is therefore to dissect out which of the various
features expressed in a cell-cycle-dependent manner
are important for preferential cell allocation.
Exposure to medium low in calcium is a procedure
that readily and reversibly inhibits intercellular flattening but does not reverse either gap junctional
formation (Goodall, 1986) or the various features
characterizing the polarized state (Handyside, 1980;
Johnson & Maro, 1984; Maro, Johnson, Pickering &
Louvard, 1985). We therefore took 2/8 pairs of cells,
all of which were in the latter (post-7 h) half of the
fourth cell cycle and all of which had therefore
completed intercellular flattening. We reversed the
flattening of these cells upon each other by exposure
to medium low in calcium, aggregated four pairs
together to make asynchronous aggregates and then
allowed cells to reflatten synchronously upon each
other, thereby removing any flattening advantage of
the older cells (as confirmed visually). As a result, the
older cells no longer contributed preferentially to the
inside cell population at the 16-cell stage. These
results therefore support the hypothesis that the
preferential contribution by early-dividing cells to the
ICM arises from the fact that they are also earlyflattening cells (Graham & Lehtonen, 1979). The
clear implication is that intercellular flattening influences the orientation of the cleavage plane.
Cell interaction is known to influence the orientation of the cleavage plane in polar 1/16 blastomeres
(Johnson & Ziomek, 1983), and recent evidence
suggests that this property is used in the embryo to
regulate the ratio of inside to outside cells at the 16- to
32-cell transition (Fleming, 1987). In contrast, a
comparison of the frequency of differentiative divisions in polar 1/8 cells dividing in isolation with that
in polar 1/8 cells dividing in a 2/8 pair does not reveal
any very striking difference (unpublished observations by S. J. Pickering, B. Maro, M. H. Johnson &
J. Skepper). It seems likely therefore that the clear
effect of order of flattening on the incidence of
differentiative divisions reported here requires multiple cell packing rather than simple contact per se.
Fig. 3. Schematic outline of the two types of mechanism
by which cell interaction might modify the orientation of
cleavage planes. Cells in isolation (A) are shown dividing
randomly. In B, cell contacts influence the position at
which the spindle forms, favouring either pole or equator
formation in the vicinity of the contact point. In C, it is
cell shape that is influenced by the cell interaction,
determining cleavage plane orientation as a result of
simple geometric considerations.
There are two types of mechanism whereby an
intercellular interaction might influence cleavage
planes. Cell interaction might lead to a 'marking' of
the internal face of the cytocortex adjacent to the
contact point and this marked area could then act as a
focus to orient either a pole or the edge of the
equatorial plate of the developing spindle (Fig. 3B;
Gunning, 1982; Palevitz, 1986). Alternatively, cell
flattening could influence cell shape and shape
changes could secondarily determine the long axis of
the spindle with respect to the polar axis of the cell,
thereby effectively determining the orientation of the
Division order and cell allocation
cleavage plane (Fig. 3C; Meshcheryakov, 1978; Freeman, 1983). In the 8-cell mouse embryo, it seems
unlikely that intercellular contact influences directly
the orientation in which the nascent spindle is set up
(Fig. 3B). Rather, the effect of cell packing on cell
shape forces a higher differentiative division rate in
early flattening cells (Fig. 3C). It seems likely that
such an influence of packing on cell shape is also
responsible for regulating cleavage plane orientation
at the 16- to 32-cell transition (Johnson & Ziomek,
1983).
We wish to acknowledge the support and stimulation of
Tom Fleming, Gin Flach, Brendan Doe and Simon Hanna.
This work was supported by giants from Trinity College,
Cambridge and the H. E. Durham Fund of Kings' College,
Cambridge to C.L.G., and by grants from the Medical
Research Council and the Cancer Research Campaign to
M.H.J.
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