/. Embryol. exp. Morph. Vol. 48, pp. 37-51, 1978
Printed in Great Britain © Company of Biologists Limited 1978
37
Cell division and cell allocation
in early mouse development
By S. J. KELLY, 1 J. G. MULNARD 2 AND C. F. GRAHAM, 13
From the Department of Zoology, Oxford
SUMMARY
Cell division was observed in intact and dissociated mouse embryos between the 2-cell
stage and the blastocyst in embryos developing in culture. Division to the 4-cell stage was
usually asynchronous. The first cell to divide to the 4-cell stage produced descendants which
tended to divide ahead of those cells produced by its slow partner at all subsequent stages of
development up to the blastocyte stage. The descendants of the first cell to divide to the
4-cell stage did not subsequently have short cell cycles. The first cell or last cell to divide
from the 4-cell stage was labelled with tritiated thymidine. The embryo was reassembled,
and it was found that the first pair of cells to reach the 8-cell stage contributed disproportionately more descendants to the ICM when compared with the last cell to divide to the 8-cell
stage.
INTRODUCTION
The cells of the preimplantation mouse embryo do not divide in synchrony
with each other and this raises the possibility that physiological differences
exist between the cells of the embryo (e.g. Dalcq, 1957; Mulnard, 1967;
Borghese & Cassini, 1963; Lewis & Wright, 1935). We have investigated division
asynchrony and the relationship between this asynchrony and the allocation of
cells to the inner cell mass (ICM) and to the trophectoderm of the blastocyst.
MATERIALS AND METHODS
Supply and culture of embryos
The embryos were obtained from natural matings of a variety of strains which
are indicated in the figure legends. The embryos were dissected into prewarmed,
pre-equilibrated Whitten's (1971) medium and cultured in microdrops (approximate volume 0-05 ml) in batches of paraffin oil selected for absence of toxicity
to cultured embryos (Boots Pure Drug Co., U.K.), under a humid gas mixture of 5 % CO2, 5 %O2, and 90 %N2 at 37 °C. The zonae pellucidae were
removed with pronase (Calbiochem, U.K., technique of Mintz, 1967). In some
1
Authors' address: Department of Zoology, South Parks Road, Oxford, 0X1 3PS, U.K.
Author's address: Laboratoire d'Anatomie et d'Embryologie Humaines, Rue aux Laines
97, 1000 Bruxelles, Belgium.
3
Reprint requests to C. F. Graham.
2
38
S. J. KELLY, J. G. MULNARD AND C. F. GRAHAM
experiments the cells of the embryo were dissociated by gentle pipetting through
a flame polished micropipette. Dissociation was sometimes assisted by placing
the embryos in Whitten's medium in which the calcium concentration had been
lowered to 0-02 mM.
Zona-free eggs were cultured either in siliconized glass dishes (siliconized
with Repelcote, BDH, U.K.) or in bacteriological grade plastic dishes (Sterilin
Ltd., Richmond, U.K.). Eggs with the zona intact were also cultured in plastic
tissue culture dishes and flasks (the latter were 25 cm2 tissue culture flasks,
Falcon Plastics, Oxnard, California). For prolonged observations on the microscope stage it was helpful to culture the embryos in sealed flasks to maintain
the correct gas mixture ('embryo in bottle' technique). In almost all cases
a channelled microscope stage was warmed by water from a thermocirculator
(Churchill Ltd., Greenford, Middlesex, U.K.).
Cine films were made of embryos cultured in a different medium under
different conditions (Mulnard, 1967). Data obtained with this method is
indicated in the figure legends.
Observations of division asynchrony
The cells were observed at regular intervals (see Table legends). Complete
embryos (zona on or off) were drawn by eye to record the three-dimensional
arrangement of cells. In some cases the embryos were drawn and also photographed with a still camera. The photographic records were useful if the level
of focus was recorded. Embryos tend to roll about when contained inside a zona
and to avoid confusion, one cell was frequently marked with an oil droplet. The
oil was silicone fluid (MS 550, BDH, U.K.), and the injection procedure and
drop size were as described by Wilson, Bolton & Cuttler (1972) with some
minor changes (Graham & Deussen, 1978). Oil droplets were not required to
follow cells in the cine film records.
To observe cell division in dissociated embryos dividing to the 16-cell stage,
it was necessary to lower the Ca2+concentration to 0-04 mM just before the
16-cell stage. This medium reduces adhesion between the cells and cell outlines
remain clear.
Data on division asynchrony was rejected if the cells did not divide through
two further divisions after the division at which the data was collected (about
5 % of the embryos were rejected). Usually both dissociated and intact embryos
developed well up to the blastocyst stage (see cell numbers in Table 2). Oil
injected cells either lysed in the first hour after injection (about 10 % of the cells)
or divided as well as controls up to the blastocyst stage. At this stage
trophectoderm cells which contained an oil droplet tended to lyse if the embryo
was inside the zona pellucida.
Cell allocation and cell division
39
Labelling of cells and reassembly of the embryo
The experiments in section 3 of this paper were conducted on embryos which
had been dissociated at the 4- to 7-cell stage. The division of each of the four
cells to the 8-cell stage was observed at 30 min intervals. Either the first or the
last cell to divide to the 8-cell stage was selected and the pair of daughter cells
were immediately labelled for 2 h in [3H]thymidine (specific activity 26 Ci/mM,
Radiochemical Centre, Amersham, U.K., technique described by Kelly &
Rossant, 1976). They were rinsed in several changes of a 1:1 mixture of Whitten's
medium: heat inactivated foetal calf serum during the next hour (Flow Laboratories, Irvine, Scotland). Subsequently they were cultured for 3-5 h in the
serum containing medium and then rinsed in Whitten's medium before
reassembly. The unlabelled cells were treated similarly. The pairs of cells from
each embryo were arranged in two sets of four (Fig. 1 A), and then one set was
placed above the other so that each cell made contact with three other cells and
so that the centre of each cell was near the corner of a cube (Fig. 1 B). The
embryos were then cultured to the blastocyst stage (32-42 h). They were fixed,
embedded, and sectioned at 4^m (techniques from Hillman, Sherman &
Graham, 1972). Line drawings or photographs were made of each section of each
blastocyst and the cell numbers in the ICM and the trophectoderm counted.
The sections were processed for autoradiography and the autoradiographs were
exposed for 2 weeks (Hillman et al. 1972). The numbers and locations of
labelled cells were scored. Previously it has been shown that tritiated thymidine
does not prevent cells at a similar stage of mouse development from either
dividing or differentiating (Kelly & Rossant, 1976).
RESULTS
The results are arranged in three sections. First, there is data which shows
that division asynchrony is a common feature of preimplantation mouse
development. Second, there is data which shows that the first cell to divide to
the 4-cell stage within each embryo tends to produce daughter cells which
divide ahead of the daughters of the second cell to divide to the 4-cell stage.
Lastly, there is evidence that the first cell to divide to the 8-cell stage contributes more daughter cells to the ICM than does the last cell to divide to the
8-cell stage.
1. Asynchrony of cell division within the embryo
Asynchrony of cell division within the embryo was usually obseived between
the 2- to 4-, 4- to 8-, and 8- to 16-cell stages (Table 1). In this Table, data from
a variety of mouse strains were combined because there was no obvious difference between the strains. Data on division in intact embryos was collected in
two ways. With long intervals of observations (5-20 min), 4 out of 28 appeared
to divide synchronously; this synchrony was probably an artifact of the long
40
S. J. KELLY, J. G. MULNARD AND C. F. GRAHAM
Table 1. Asynchrony of cell division within the embryo
Observations on embryos from C3H/H, (C57BL6x CBA) F2, A2G, and 129J/Sv.
Asterisk marks the cine film observations on the embryos of Swiss albino mice.
These embryos were observed at 40 sec intervals. The times of division do not
exactly correspond to the intervals of observation. This is because cells were followed
continuously under the microscope when the start of cytokinesis was noticed, a
indicates that the AB cells (the first to divide) had a significantly shorter 2-cell-stage
cell cycle than the CD cells (the second to divide) at the 0-1 % level; b indicates that
it was shorter at the 1 % level (related t test, Meddis, 1975).
Division
2-A
Stage at which
culture initiated
Intact 2-cell
Intact 2-cell
Intact 2-cell
Zona free 2-cell
Zona free 2-cell
4-8
Zona free 2-cell
Intact 2-cell
Intact 2-cell
Zona free 2-cell
Zona free 2-cell
8-16
Zona free 2-cell
Intact 4-cell
Zona free 4-cell
Zona free 2-cell
Treatment
None
None
Oil in one
cell
None
Oil in one
cell
Dissociated
None
Oil in one
cell
None
Oil in one
cell
Dissociated
None
Dissociated
Dissociated
twice
Number
asynchronous/
total
number
Interval from first
to last division
Obser- within asynchronous
vation
embryos (min)
-A
^
interval i
(min)
Mean
Range
12/14
5-20
12/12
12/14
54 a
68 a
5-20
44a
5/5
14-115
1-180
5-109
8-87
2-113
5
52
10/12
5-20
49 b
14/16
3/3
9/9
5-15
5-30
5-30
143
108
15-157
60-240
30-180
6/6
4/4
5-20
5-20
223
178
68-315
26-309
30
111
131
87
149
30-180
18-320
15-225
50-246
15/15
13/13
8/8
7/7
5-10
5-15
5-20
56 b
observation intervals. For when data was collected from cine film (every 40 sec),
12 out of 12 embryos divided asynchronously; one embryo in this series had
a division interval of 1 min (the time between completion of division in the first
and last cell).
For the 4- to 8- and 8- to 16-cell divisions, all the embryos displayed
asynchronous divisions. The mean and range of the time intervals between
first and last divisions are given in Table 1.
The results show that the first cell to divide to the 4-cell stage within each
embryo usually had a shorter 2-cell-stage cell cycle than its slower partner
(statistically significant division asynchrony, see legend to Table 1). The data
also showed that neither the various treatments nor the time at which culture was
initiated had a marked or consistent effect on the asynchrony which was
Cell allocation and cell division
41
observed at the 2- to 4- and 4- to 8-cell divisions. It is therefore likely that the
asynchrony which was observed at the 8- to 16-cell division (in embryos dissociated at the 2-cell stage and again after the next division) resembled that
which occurred in intact embryos developing in culture.
Clearly asynchrony of cell division is a regular feature of preimplantation
mouse development.
2. Division order and cell cycle durations
Division order was observed within the embryos to find out whether the first
cell to divide from the 2- to 4-cell stage (nominated the AB cell) produced
daughter cells that divided ahead of the daughters of its slower partner
(designated the CD cell, see Gulyas, 1975). The data from various strains were
combined because no interstrain differences were observed.
2- to 8-cell divisions
The division order to the 8-cell stage is shown in Table 2A. Notice that an
AB daughter was usually the first cell to divide to the 8-cell stage (40/48 cases),
and in no case did CD produce the two first cells to divide to the 8-cell stage.
This pattern of cell division was not disturbed by removal of the zona pellucida,
by injection of oil droplets, or by dissociation of the embryo at the 2-cell stage.
This last observation suggests that this pattern of division is a property of the
individual cells rather than the result of interactions between all the cells of the
embryo.
The duration of cell cycles between the 4-cell and 8-cell stages was studied
next in an attempt to account for the observed division order. The cell cycle
durations of the daughters of AB and of CD are shown in Table 3. This Table
contains data from a variety of strains which were exposed to diverse treatments.
Nevertheless, the means and trends were similar for all treatments and the data
were combined for analysis by the related t test (Meddis, 1975). This test
compares the differences in cell cycle duration within each embryo. Several trends
emerge from these comparisons. The faster daughter of AB had a significantly
shorter 4-cell stage cell cycle than the slower daughter of AB (significantly
different at the 0-1 % level). However, the cell cycle of the faster daughter of
AB was just longer than that of the faster CD daughters (significantly different
at the 1 %).
The cell cycle durations of the faster and slower daughters of CD were not
significantly different from each other.
Notice that the mean cell cycle of the AB daughters was longer than the mean
cell cycle of the CD daughters (significantly different at the 1 % level). The
implication is that AB daughters tend to be first to the 8-cell stage simply because
they were derived from the cell which was ahead at the 2- to 4-cell division, and
not as a result of an intrinsically shorter cell cycle after the 2-cell stage.
42
S. J. KELLY, J. G. MULNARD AND C. F. GRAHAM
Table 2. Tendency of AB daughters to divide ahead of CD daughters
The embryos used in these experiments were selected for asynchrony at the 2- to 4cell division using intervals of observation ranging from 5 to 30 min. The cell
divisions in section A (to the 8-cell stage) could be observed in normal culture
medium. To observe the cell divisions from the 8- to the 16-cell stage (section B),
it was necessary to lower the calcium content of the medium to 004mM and no
compensation was made for the slight change in osmolarity. To observe the cell
numbers in section C, the embryos were flattened out and stained according to the
procedure of Tarkowski (1966). The asterisk draws attention to the cell numbers in
the blastocysts derived from AB and CD. These numbers were significantly different
at the 25 % level by the related t test (Meddis, 1975).
Frequently two cells had divided to a particular cell stage during an interval in
observation. Data from these observations are excluded and this exclusion accounts
for the variation in the number of embryos along particular lines of Section A of this
Table.
The embryos in these experiments were from the same strains as those listed in the
legend to Table 1.
Number
of
Treatment
Zona on
Zona on, oil in one cell
Zona off
Zona off, oil in one cell
Zona off, dissociated
Totals
embryos
Mean (range) One AB
interval from daughter
2- to 4-cell first to the
stage
8-cell stage
A. Development to the 8-cell stage
17
63-5
11/15
(1-180)
4
44-7
3/3
(5-135)
7
66-3
5/5
(16-120)
11
53-7
6/6
(18-125)
52-3
26
15/19
(30-157)
65
—
40/48
Both AB
daughters
to 8-cell
One AB
stage
daughter
before
last to the
CorD
8-cell stage
5/10
6/17
1/4
2/4
3/7
2/7
3/10
2/9
12/23
6/26
24/54
18/63
B. Development to the 16-cell stage (sequential observations> on nine embryos)
Treatment
Zona off,
dissociated
Mean
(range)
interval
from
?. tn 4-rell
stage
51-3
(15-105)
Frequency with which an AB daughter divided to form an
embryo with one of the following cell numbers
A.
9
10
11
12
13
14
15
16
7/9
6/9
8/9
6/9
4/9
2/9
1/9
2/9
43
Cell allocation and cell division
Table 2 (cont.)
C. Development to morula and blastocyst
Treatment
Morula - Zona off,
dissociated
Blastocyst - Zona off,
dissociated
Mean (range)
interval from
1. tr\ 4-reil
stage
57-3
(30-75)
84-4
(30-210)
Frequency
with which
ABhad
formed more
Mean (range) cell number
Hoi loVifpr
U C l U g l I IW.L
cells than CD AB daughters
14-5
5/6
(12-17)
13/16
33-7
(22-54)
A
CD daughters
116
(9-15)
*
31-2
(21-42)
4- to 16-cell divisions
The division order of AB and CD descendants was investigated during the
divisions to the 16-cell stage (Table 2B). These observations were made on
embryos dissociated at the two-cell stage and again after the next division. This
procedure reduced the chances of muddling the cells. The AB descendants were
usually the first to divide to the 16-cell stage. In these nine embryos, of the first
36 cells to divide (i.e. the first four in each embryo to bring them all to the
12-cell stage, see Table 2B), 27 were AB descendants. In contrast AB descendants only contribute nine cells amongst the latter 36 cells to divide through to the
16-cell stage.
In order to compare statistically this apparent tendency for the AB descendants to reach the 16-cell stage before the CD descendants, a score of eight was
allotted to the first cell to divide in each embryo. The second was allotted a score
of seven and so on down to a score of one for the last cell to divide. Using
these scores, the Wilcoxon matched pairs test (Meddis, 1975) showed that the
AB descendants had a significantly higher score than the CD descendants
(significantly at the 5 % level T = 5). This shows that the AB descendants have
a statistically significant tendency to reach the 16-cell stage before the CD
descendants.
The duration of the cell cycles was now studied in the hope that these division
orders could be explained. Accurate data were only available for seven of these
embryos and these are in Table 3 B. This data shows that the mean time taken
for the completion of the 4- and 8-cell stages was not significantly
different for the AB and CD descendants (1475-4 and 1441-9) or for the fastest
AB and the fastest CD descendants (1435-0 and 1379-3). Again, the implication
is that the AB descendants tend to reach the 16-cell stage ahead of the CD
descendants simply because they were derived from a cell which was ahead at
the 2- to 4-cell division, and not as a result of an intrinsically shorter cell cycle
of their own. However, the durations of this period were variable amongst
the AB descendants; within each embryo the fastest AB descendant had a
44
S. J. KELLY, J. G. MULNARD AND C. F. GRAHAM
Table 3. Durations of cell cycles
A. Duration of the 4-cell stage
Mean
(range)
interval
Mean (range) duration of 4-cell stagt5 in min
•from
11. \ji 11
Treatment
Number 2- to 4-cell
of
stage
embryos
(min)
Zona on
5
Zona on, oil in
one cell
Zona off
4
Zona off, oil
in one cell
Zona off,
dissociated
6
Means
A
f
4
7
(n = 26)
CD daughters
AB daughters
A
Faster
Slower
52-4
(18-110)
42-3
(5-185)
69-7
(16-111)
78-8
(3-168)
47-4
(15-105)
766-2
(695-850)
774-7
(752-787)
7920
(765-826)
788-5
(748-840)
748-6
(710-875)
864-4
(759-1045)
835-7
(777-871)
858-7
(778-901)
901-6
(755-1202)
769-3
(720-875)
58-3
771-9
842-1
Faster
Slower
741-6
(649-825)
7710
(750-792)
766-5
(700-846)
785
(703-878)
737-3
(610-855 )
787-6
(649-910)
811-3
(755-922)
8120
(733-877)
827-6
(706-978)
7861
(615-870)
758-8
8070
803-8
781-3
B. Duration of the 4- and 8-cell cycles combined
(Observations on seven embryos, dissociated at the 2- and the 4-cell stages. Mean
interval 2- to 4-cell stage was 37-3 min, range was 1-75 min.)
Mean (range) duration in min of the 4- and 8-cell cycles
Daughters of AB (order to division)
Daughters of CD (order to division)
First
14350
(12301603)
Second
1470-7
(12751670)
Third
1479-3
(1285(1690)
A.B mean duration = 1475-4
Fourth
1516-3
(13351705)
First
Third
Second
1379-3
1412-6
1459-9
(1275(1305(13661498)
1525)
1593)
CD mean duration =: 1441-9
Fourth
1515-9
(14051601)
These observations were made on the same strains of embryos as those indicated in the
legend to Table 1.
significantly shorter 4- to 16-cell period than that of the slowest (significant at
the 1 % level). This variability was also apparent amongst the descendants of CD;
the fastest cell had a significantly shorter period than the slowest cell (significant
at the 0-1 % level). Heterogeneity in cell cycle lengths is apparently a feature
of individual cells, but does not appear to be inherited as a cell autonomous
feature of a particular cell line within the dissociated mouse embryo; this is in
contrast to the situation found in embryos with precise cell lineages (van der
Biggelaar & Boon Neemeijer, 1973).
Cell allocation and cell division
45
2-cell stage to morula and blastocyst
The division order to the morula and the blastocyst could not be directly
observed. Instead embryos were dissociated at the 2-cell stage and the cell
numbers were counted in the half size morulae and blastocysts which developed
from the AB and CD cells respectively (Table 2C). The morulae were counted
in the morning of the fouilh day of development. They had divided to form
embryos, which had they been intact, would have had a mean cell number of
26 (range 24-31). In these six embryos, the AB descendants were more numerous
than the CD descendants in five cases. The AB half and the CD half had identical
cell numbers in the sixth case.
The blastocysts were counted early on the morning of the fifth day of
development. They had divided so that, had they been intact embryos, they
would have had a mean cell number of 65 (range 49-96). In 13 out of 16 embryos,
the descendants of AB were more numerous than the descendants of CD.
Overall AB had significantly more descendants than CD (significantly different
at the 2-5 % level, related t test).
Clearly AB descendants tend to divide ahead of the descendants of CD in all
divisions from the 4-cell to the blastocyst stage. All results except the cine film
results were obtained with embryos which were observed to be asynchronous
at the 2- to 4-cell division using relatively long intervals of observation. In no
experiment were more than 30 % of the embryos discarded because of synchronous 2- to 4-cell divisions; usually less than 10% were discarded. Our
observations therefore relate to the majority of embryos in any batch.
3. Relationship between division order and cell allocation
Division order might affect the process of allocation of cells to the ICM and
to the trophectoderm of the blastocyst. This was investigated by looking for
associations between division order to the 8-cell stage and the contribution of
cells to the two tissues of the blastocyst. Embryos were dissociated into single
cells at the 4-cell stage and the division order to the 8-cell stage was observed.
Within each embryo the daughters of either the first or the last cell to divide to
the 8-cell stage were labelled with tritiated thymidine. Next the embryos were
reassembled in such a way that all the eight cells were in similar positions
relative to each other (Fig. 1, and Materials and Methods). From our previous
observations it was probable that the first cell to divide to the 8-cell stage would
be a daughter of AB and that the last cell to do so would be a daughter of CD;
our procedure therefore allowed us to follow some of the daughters of AB and
CD through to the blastocyst stage.
First it is necessary to assess the effect of the labelling procedure on the results.
Since two cells were labelled at the 8-cell stage, these should divide to form 25 %
of the cells in the blastocyst. Tables 4 and 5 show that the mean percentage of
labelled cells was less than expected - 21 % (significantly different a t P = 0-001
4
EMB 48
46
S. J. K E L L Y , J. G. M U L N A R D A N D C. F. G R A H A M
Fig. 1. Re-assembly of labelled embryos. All the cells from a dissociated 4-cell
embryo were kept apart and divided to the 8-cell stage. The pairs of cells were
arranged in two sets of four (Figure 1 A). When these sets had adhered, one set was
placed on top of the other (Figure 1B). Scale bar = 50 /*m.
Table 4. Labelled and unlabelled cell counts of blastocysts in which the progeny
of the first cell to cleave away from the 4-cell stage were labelled for 2 h in
[3H]thymidine
Labelled cells
Embryo Total
no.
cell no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean
Ranges
43
51
52
57
59
67
67
67
69
72
73
79
80
81
86
No. in
ICM ICM/total
14
15
18
15
16
19
13
20
19
22
19
21
19
22
15
0-326
0-294
0-346
0-263
0-271
0-283
0194
0-299
0-275
0-306
0-260
0-266
0-238
0-272
0174
0-272
0-1740-346
r "
Total
ICM
ICM/total
10
8
11
13
14
13
10
12
13
17
17
15
18
18
17
3
2
4
3
4
3
5
4
4
8
4
6
6
6
0-300
0-250
0-364
0-231
0-286
0-231
0-500
0-333
0-308
0-471
0-235
0-400
0-333
0-333
0059
0-309
t
0-0590-500
No.
labelled
Total no.
in
labelled/ ICM/total
total no. no. in ICM
0-233
0157
0-212
0-228
0-237
0194
0149
0179
0188
0-236
0-233
0190
0-225
0-222
0198
0-205
0-1570-237
0-214
0133
0-222
0-200
0-250
0158
0-385
0-200
0-211
0-364
0-211
0-286
0-316
0-273
0067
0-233
0-0670-385
Data obtained on mice from the PO and C57BL6 strains. Comparisons were made of
the differences between the figures appearing in columns 8 and 7 and columns 6 and 3
(see text), and those obtained between the same columns in Table 5.
Blastocysts have been arranged in order of total cell number.
47
Cell allocation and cell division
Table 5. Labelled and unlabelled cell counts of' blastocysts in which the progeny
of the last cell to cleave away from the 4-cell stage were labelled for 2 h in
[3H]thymidine
Labelled cells
Embryo
no.
Total
cell no.
1
2
3
4
5
6
7
8
9
Means
Ranges
36
50
57
64
66
68
72
74
85
No. in
1CM
10
14
12
16
17
18
16
12
24
A
ICM/total
Total
0-278
0-280
0-211
0-250
0-258
0-265
0-222
0162
0-282
0-245
1880-282
7
12
14
14
17
17
18
15
6
ICM ICM/total
0
4
0
3
4
2
6
2
1
0000
0-333
0000
0-214
0-235
0118
0-333
0133
0167
0170
0-0000-333
No.
labelled
Total no.
in
labelled/ ICM/total
total no. no. in ICM
0194
0-240
0-246
0-219
0-258
0-250
0-250
0-203
0071
0-215
0-0710-258
0000
0-286
0000
0188
0-235
0-111
0-375
0167
0042
0156
0-0000-375
Data obtained on mice from the PO and C57BL6 strains. Comparisons were made as
described in the legend to Table 4.
The blastocysts have been arranged in order of total cell number.
for 23 D.F., Student's t test, Bailey, 1959). Tritiated thymidine appears to slow
the rate of cell division and might therefore obscure the phenomenon under
investigation. The degree of retardation, however, was similar when either the
first or the last pair of cells to the 8-cell stage were labelled (20-5 % does
not differ significantly from 21-5%). It was subsequently assumed that any
effect of the tritiated thymidine on cell allocation would be the same in both
series of experiments and that it was legitimate to look for differences between
the series.
The distribution of labelled cells in blastocysts formed from embryos in which
the daughters of the first cell to divide to the 8-cell stage were labelled is given in
Table 4, and that for the blastocysts formed from embryos in which the
daughters of the last cell to divide to the 8-cell stage were labelled is given in
Table 5. These tables also show the calculated proportions used in the analyses
of the distribution of the labelled cells. If the labelled cells were distributed
randomly in the blastocyst, then the proportion of labelled cells amongst the
total number of cells in the ICM (column 8) should be the same as the proportion
of the labelled cells in the blastocyst as a whole (column 7) (comparison 1)
Another way of looking at the distribution of labelled cells is to compare the
proportion of the total number of labelled cells which appear in the ICM
(column 6) to the proportion of the total cell number that appear in the ICM
(column 3) (second comparison); this comparison makes some allowance for
4-2
48
S. J. KELLY, J. G. MULNARD AND C. F. GRAHAM
the individual variation in the size of the ICM. Deviations from a random
distribution will produce differences between these proportions in each case.
If the progeny of the first cell to cleave away from the 4-cell stage have a significantly increased chance of appealing in the ICM over that of the progeny
of the last cell to cleave away from the 4-cell stage, then the differences between
these proportions in the first series should differ significantly from the differences between them in the second series. These differences were compared by
performing two sample t tests (Bailey, 1959) on the two comparisons in each
series. (These tests were legitimate because the variances of the two samples
were similar in each series). The test showed that for both comparisons there
was a statistically significantly greater contribution of the progeny of the first
cell to divide away from the 4-cell stage to the ICM than of the progeny of the
last cell to divide (significant at P = 0 1 for the first comparison and at
P = 0-05 for the second).
Clearly there is an association between division order and the contribution
of cells to the ICM. Notice that there is no evidence that the first cell to divide
to the 8-cell stage has formed more daughters than the last cell to divide to the
8-cell stage. This is possibly because the blastocysts were fixed at various times
without any attempt to match the two series.
DISCUSSION
AB and CD cell division
Chance processes could account for the observation that one cell of the 2-cell
embryo divides to the 4-cell stage before the other; this is probably not the case
because we have noticed that there is some regularity in the orientation of the
cell which divided first (unpublished observations).
Lewis & Wright (1935) believed that the first cell to divide was the larger
cell produced by unequal first cleavage. This relationship was not noticed in this
work (see also Mulnard, 1967) although the first cell to divide appeared to
increase in size just before division. Early in the 2-cell stage, the two cells do in
fact differ slightly in dry mass and this difference increases a little during interphase (Abramczuk & Sawicki, 1974). There are other signs that the two cells
are metabolically distinct: the nucleoli may differ in the time at which they
acquire staining properties which are thought to indicate the presence of
ribosomal RNA synthesis (Engel, Zenzes & Schmid, 1977). and they may also
differ in the duration of DNA synthesis (Luthardt & Donahue, 1975). Possibly
the metabolism of the two cells becomes different during the 2-cell stage.
It appears that the AB cells do not transmit to their daughters the character
of short cell cycles when they are grown in isolation from the CD cells. Thus
AB daughters do not have a shorter cell cycle than CD daughters at the 4-cell
stage. Similarly the period from the 4- to the 16-cell stage is not shorter for AB
daughters; in fact it is slightly longer. The observed regularity of division order
Cell allocation and cell division
49
during development then appears to be the consequence of two processes: there
is the initial asynchrony at the 2- to 4-cell division followed by similar mean cell
cycle times for AB and CD descendants, which do not obscure this asynchrony.
There is therefore no evidence for a transmissible state which segregates at the
2-cell stage of development. We do not have sufficient evidence to decide if such
a state segregates at the 4-cell stage; cell cycle heterogeneity is observed at all
stages. Our observations on the division order to the 8-cell stages confirm
previous suggestions by Lewis & Wright (1935) and Borghese & Cassini (1963).
A (locations of cells to the ICM and to the trophectoderm
Previously it had been noticed that the first cells to divide to the 16-cell stage
tended to form inside cells in intact cultured embryos (Barlow, Owen &
Graham, 1972). It follows from our data on division order that these cells were
usually the products of AB.
The labelling experiments in this paper show that within an embryo, the first
cell to divide to the 8-cell stage tends to contribute more cells to the ICM than
does the last cell to divide the 8-cell stage. Since the first cell to divide to the
8-cell stage is usually derived from AB (in 40/48 cases), it is likely that one
4-cell-stage daughter of AB contributes disproportionately more cells to the
ICM than does one 4-cell-stage daughter of CD.
Our experimental procedure may obscure an even more regular pattern in the
intact embryo. First, our observations were made on embryos which were
dissociated at the 4-cell stage and reassembled at the 8-cell stage. Any relationship between division order and contribution to the ICM which depends on
mechanisms which operate before reassembly of the embryo was therefore
excluded. There is for instance a relationship between division order and cell
position in the intact embryo at the 8-cell stage (Graham & Duessen, 1978).
Second, we did not follow the daughters of AB and CD separately through from
the 2-cell stage in these labelling experiments, and some of the daughters of CD
may have been amongst the first cells to the 8-cell stage. Third, the labelling
procedure slowed the division of the labelled cells. Fourth, the cell arrangements
in the reassembled embryos were different from those in intact embryos where
the daughters of each cell at the 4-cell stage are in different relative positions;
in the reassembled embryos the cells were in identical positions relative to each
other. Despite all these difficulties, our results demonstrate that there is
a relationship between division order to the 8-cell stage and the contribution of
of cells to the ICM. This relationship must depend in these experiments solely
on mechanisms which operate after the reassembly of the embryos.
In the reassembled embryos, the first and the last cell to divide to the 8-cell
stage together formed about an half of the cells in the ICM. It is likely that the
second and the third cell to divide to this stage also contributed to the ICM. Our
observations confirm the studies of Wilson et al. (1972) which showed that no
cell at the 2- and 4-cell stage contributed exclusively to the ICM in intact
50
S. J. KELLY, J. G. MULNARD AND C. F. GRAHAM
embryos. Each cell of a 4-cell embryo has previously been shown to be able to
form all the tissues of an adult mouse (Kelly, 1977), and our results suggest that
each cell of a 4-cell embryo contributes to the 1CM in these reassembled embryos.
CONCLUSIONS
1. One of the cells in the 2-cell embryo is the first to divide to the 4-cell stage
(AB cell) and its daughters tend to divide ahead of those cells derived from its
slower partner at all subsequent stages of development up to the blastocyst
stage.
2. AB descendants do not have shorter cell cycles than the other cells of the
embryo during subsequent stages of development.
3. The first cell to divide to the 8-cell stage tends to contribute disproportionately more descendants to the 1CM when compared to the last cell to divide to
the 8-cell stage.
We would like to thank A. J. Copp, J. Haywood, R. L. Gardner, V. E. Papaioannou and
J. West for helpful discussions. The MRC kindly supported these studies.
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(Received 16 February 1978)