Download Mechanism of size regulation in mouse embryo aggregates

J. Embryol exp. Morph. Vol. 72, pp. 169-181,1982
Printed in Great Britain © Company of Biologists Limited 1982
169
Mechanism of size regulation in mouse embryo
aggregates
By N. E. LEWIS, AND J. ROSSANT 1
From the Department of Biological Sciences, Brock University,
St Catharines, Ontario
SUMMARY
A detailed comparison of the postimplantation development of normal and double-sized
mouse embryos, produced by aggregating two 8-cell stage eggs, revealed that size regulation
occurred in the double embryos between 5 days, 16 h post coitum (p.c.) and 6 days, 16 hp.c.
Size regulation occurred simultaneously in all tissues, suggesting that a single regulatory
mechanism may control size in the early embryo. Size regulation appeared to be brought
about by alteration in cell cycle length. There was no obvious increase in cell death in the
double embryos nor an increase in the non-dividing cell population. However, colcemid
treatment revealed a significant difference in mitotic index between double and control
embryos over the period of size regulation. Control embryos showed a proliferative burst
around 6 days, 8 h p.c. which did not occur in the double embryos. It is not yet clear whether
this control of proliferative activity in double embryos is exerted by the embryo itself or by
the uterine environment.
Histological analysis also suggested that proamniotic cavity formation, which occurs before
size regulation, was dependent on total cell number and not on the number of cell cycles
undergone since fertilization. Proamniotic cavity formation was observed to occur at different
times but at similar cell numbers in double, control and half embryos.
INTRODUCTION
Genetic chimaeras produced by aggregating early embryos have proved very
useful for studying development and differentiation in the mouse (Mintz, 1974;
McLaren, 1976), and have also provided insights into embryonic growth regulation. Aggregates of two (Tarkowski, 1961; Mintz, 1962), three (Markert &
Petters, 1978) or four embryos (Petters & Markert, 1980) all produce viable
offspring of normal size, indicating that there are growth control mechanisms
in the embryo which can compensate for increased preimplantation size. Buehr
& McLaren (1974) presented evidence that size regulation occurred in double
embryo aggregates shortly after implantation around the time of proamniotic
cavity formation. However, no attempt was made to determine mechanisms
responsible for such regulation. The aim of the present study was to use serial
reconstructions of double embryo aggregates to:
(1) pinpoint clearly the period of time over which size regulation occurred,
1
Author's address for reprints: Department of Biological Sciences, Brock University, St
Catharines, Ontario, L2S 3A1, Canada.
170
N. E. LEWIS AND J. ROSSANT
(2) determine whether a single control mechanism could account for size
regulation or whether tissues regulated size independently.,
(3) determine the most likely mechanism of size regulation.
Results of the study indicated that size regulation occurred in all tissues at the
same time and was accomplished by an alteration in cell cycle length.
MATERIALS AND METHODS
Mouse strains and culture media used
All embryos were obtained from natural matings of random-bred Ha(ICR)
mice. PB1 medium (Whittingham & Wales, 1969) supplemented with 10%
foetal calf serum (FCS) was used for recovery, manipulation and transfer of
embryos. Embryos were cultured overnight in a-modified MEM (GIBCO) +
10% FCS at 37 °C in an atmosphere of 5 % CO2 in air.
Production of double embryos
Eight-cell embryos were flushed from the oviducts of pregnant females between 12.00 and 15.00 h on the second day following vaginal plug formation.
Zonae pellucidae were removed by a brief incubation in acidified Tyrode's
solution (pH 2-5) (Nicolson, Yanagimachi & Yanagimachi, 1975). Embryos
were washed and transferred singly or in pairs to drops of a-MEM under light
liquid paraffin in Falcon bacteriological petri dishes. Double embryo aggregates
were made by gently pushing two uncompacted 8-cell embryos together. Once
aggregation had begun, the culture dish was placed in the incubator and cultured
overnight. The following afternoon, well-integrated aggregates and control
embryos had formed late compacted morulae or early blastocysts. Five double
embryos were transferred to one uterine horn of each recipient female on the
second day after vaginal plug formation. Five control single embryos were
transferred to the opposite horn. In all further analysis embryos were staged by
the time postcoitum of the pseudopregnant recipient rather than by the age of
the embryos. Mating was assumed to occur at the midpoint of the dark period
(2.00 a.m.).
Processing of embryos
(a) Untreated
Recipients carrying double and control embryos were killed every 8 hours
from 4 days, 0 h to 6 days, 16 hpost coitum (p.c). Uteri were dissected and fixed
overnight in AFA fixative (acetic acid: formalin: alcohol: water; 1:1:3:5). After
dehydration and embedding in paraffin wax, sections were cut at 7 ju,m, mounted
and stained with haematoxylin and eosin.
(b) Labelled with [3H]thymidine
Recipient females carrying both double and control embryos were injected
with [3H]thymidine and killed at 5 days, 22 h and 6 days, 2 h p.c. Females were
Mechanism of size regulation in mouse embryo aggregates 171
given four intraperitoneal injections of 50/tCi a-methyl[3H]thymidine (Amersham, specific activity, 20 /*Ci/mmole) at hourly intervals and were killed 1 h
after the last injection (Solter, Skreb & Damjanov, 1971). Uteri containing
decidual swellings were fixed and processed as before. Wax sections were
mounted on glass slides, taken down to water and treated with 10% trichloroacetic acid for 10 min. The slides were then dipped in Kodak NTB3 nuclear
track emulsion, exposed for four weeks at 4 °C in the dark and then developed,
fixed, and stained with haematoxylin and eosin.
(c) Colcemid-treated
Several recipient females were given a single intraperitoneal injection of 0-6 ml
of a 0-1 mg/ml solution of demecolcine (Sigma) in phosphate-buffered saline
two hours prior to killing (Copp, 1978). These recipients were killed every 8 h
between 5 days, 16 h and 7 days, 0 h p.c. Uteri containing decidua were fixed
and processed as described for untreated embryos.
Analysis of sectioned material
Camera-lucida drawings were made of all sections which contained embryonic
material. The boundaries of tissue types and all mitotic figures were recorded
on the drawings. Cell number, mitotic index and labelling index were calculated
for the various tissues. Figure 1 illustrates which tissues were included in cell
number estimates at different stages. In the preimplantation embryo, total cell
numbers of trophectoderm and inner cell mass (ICM) were calculated, but, in
the postimplantation embryo, trophoblast giant cells plus adjacent parietal
endoderm and ectoplacental cone were excluded because it was difficult to
discern the boundaries between these tissues and maternal tissues. Extraembryonic ectoderm was the only postimplantation trophectoderm derivative
included in the calculations.
(a) Cell number estimates
Direct counting of cell number was clearly impossible in later stage embryos
and so cell number throughout was estimated by calculating the number of
nuclei in a small area of tissue and multiplying this by the total tissue area per
embryo as estimated from planimeter measurements of the camera-lucida
drawings. Abercrombie's formula (Abercrombie, 1949) was used to estimate
number of nuclei per unit area.
Abercrombie's formula is P = AxM/L + M, where
P
A
M
L
=
=
=
=
total number of nuclei,
number of nuclear fragments,
thickness of section (/tin),
average length of nuclei (/*m).
M was known to be 7 /im. A was estimated for each tissue by obtaining the
average of several counts of nuclear fragments in different small areas (20-50
172
N. E. LEWIS AND J. ROSSANT
planimeter units). No significant difference in A could be found between ICM,
embryonic ectoderm, and extraembryonic ectoderm at different times or between single and double embryos (mean number of nuclear fragments/ten
planimeter units in all tissues except endoderm = 5-0, range 4-7-5-3). Thus, the
same value of A was used for calculating P in these tissues at all stages. However, the value of A for endoderm differed significantly from the other tissues
and also varied significantly with time (range of number of nuclear fragments/
ten planimeter units = 2-3-3-7). Thus, separate values of A, and hence separate
values of P, were used for calculating total endoderm cell number at all stages
of development. Nuclear length measurements varied little from stage to stage
or tissue to tissue (range 7-8-8-2 /im) and a mean nuclear length, L, of 8 /*m
was used for all calculations. Once a value of P per unit area was derived for
each tissue, total cell number was readily estimated by multiplying by the total
section area.
(b) Mitotic index and labelling index
The mitotic index (MI) was calculated for untreated and colcemid-treated
embryos by counting the number of complete metaphases and anaphases and
expressing them as a percentage of total cell number. The labelling index (LI)
was obtained from [3H]thymidine-treated embryos by scoring the percentage of
labelled cells in the population. Background labelling was low (> 2 grains
overlying the cytoplasm of a cell) and a cell was considered labelled if more than
three silver grains overlaid the nucleus (Copp, 1978).
RESULTS
Development of aggregates
No difference in the rate of successful development was observed between
double and control embryos. For double embryo aggregates, 39 % of all embryos
transferred to recipients that became pregnant developed successfully, while
40 % of control embryos developed after transfer. Double and control embryos
appeared morphologically normal and there was no obvious increase in the
amount of cell death in double embryos at any stage of development. Treatment
with colcemid and [3H]thymidine had no obvious deleterious effects on the
embryos. Only pregnant mice containing successful implants in both horns were
used for further analysis.
Size regulation in double embryos
Analysis of cell numbers of double and single embryos revealed that the
double embryos were at least twice as large as single embryos early in development (Table 1). Cell numbers of double and single embryos remained significantly different until 6 days, 16 h. When the ratio of cell numbers of double to
control embryos was plotted against time (Fig. 2) it was clear that the ratio
Mechanism of size regulation in mouse embryo aggregates 173
I 1 ^ * 9 4 - Endoderm
/
— Extraembryonic
ectoderm
4dl2hp.c.
1
1
— Embryonic
ectoderm
\
— Endoderm
V A
V\
V V ^ —jT
T r o p h e c t o d e r m \f/ W* i"k *W
Extraembryonic
ectoderm
)i
^ ^ - ^
Embryonic
ectoderm
Endoderm
5d 12hp.c.
6d \2hp.c.
Fig. 1. Diagram of tissues included in cell number estimates.
Table 1. Cell number and mitotic index {MI) of the total tissue
in double and control embryos
Days p.c.
4d, Oh
4d, 8h
4d, 16 h
5d, Oh
5d, 8h
5d, 16 h
6d,0h
6d, 8h
6d, 16 h
Number of
embryos
Double ControlI
7
6
10
7
6
4
7
5
7
5
11
4
8
6
7
5
5
6
Mean cell number ±S.E.
Double
Control
160±15-4
63 ±14-6
248 ±31-4
115+18-8
309 ±21-0
160±14-8
413 ±43-0
198 ±14-8
389 ±45-6
199 ±40-8
749 ±64-7
324 ±34-2
1209 ±94-2
891 ±54-8
1542 ±62-1 1216±110-8
2253 ±313-2* 2086 ±233-0*
Mean
Double
MI±S.E.
0-4 ±0-2*
1-5 + 0-5*
0-8±0-2*
l-6±0-3*
0-7±0-3*
l-0±0-2*
l-9±0-5*
0-7±0-l*
3-1 ±0-3*
Control
0-6±0-3*
0-7 ±0-4*
0-5 ±0-2*
2-2 ±0-4*
0-9±0-4*
0-7±0-3*
2-1 ±0-5*
1-1 ±0-2*
2-8±0-3*
* Denotes a non-significant difference between experimental and control values at P <
0-05 using Student's Mest.
remained around 2:1 until 5 days, 16 h. Over the following 24 h, there was a
rapid shift towards a 1:1 ratio, so that cell numbers were virtually identical at
6 days, 16 h. The Mis of all embryos were low (Table 1) and no significant
differences could be found between double and control embryos.
Analysis of cell number data for individual tissues showed that size regulation
occurred in all tissues over roughly the same time period. Both ICM and trophectoderm derivatives showed size regulation by 6 days, 16 h, with the shift
in cell numbers occurring between 5 days, 16 h and 6 days, 16 h (data not shown).
174
N. E. LEWIS AND J. ROSSANT
30,X
o
•
*
20
»
X
*
9
A
10
O
m
*
1
1
4d0h
1
1
1
4dl6h
4d8h
5d8h
5dOh
1
1
1
6d0h
6dl6h
6d8h
5 d 16 h
Time p. c.
Fig. 2. Graph of ratio of cell numbers of double and control embryos against time.
• = Total embryonic tissue; O = embryonic ectoderm; A = extraembryonic
ectoderm; x = endoderm.
When data for embryonic ectoderm, extraembryonic ectoderm and endoderm
were considered separately (Table 2), no clear differences in timing of size
regulation could be observed. A statistically non-significant difference between
cell numbers of double and control embryos occurred slightly earlier in embryonic ectoderm and extraembryonic ectoderm than in endoderm. However,
visual inspection of the graph of double: single embryo cell number ratio against
time (Fig. 2) revealed no obvious 'leading' tissue in size regulation.
Size of dividing cell population
Labelling of embryos was successfully achieved after injection of [3H]thymidine in all experiments. Both control and double embryos showed high labelling
indices in all tissues after [3H]thymidine treatment between 5 days, 18 h and
6 days, 2 h (Table 3) and there was no significant difference between single and
double embryos. Therefore, no large non-dividing and/or dying cell population
was present at the beginning of the period of size regulation in double embryos.
Mitotic activity
Embryos treated with colcemid showed cell numbers which were very similar
to those of untreated embryos and size regulation occurred at the same time
(data not shown). However, use of colcemid greatly amplified the MI and
allowed meaningful comparison of the mitotic activity of double and single
embryos. Control embryos showed a dramatic increase in mitotic activity
5d 16h
6dOh
6d8h
6 d 16 h
5 d 18hto
5d22h
5 d 22 h to
6d2h
Labelling
Period
11
4
8
6
Days p.c.
269 ±28-3
420 ±47-8*
538 ±30-0*
859 ±124-7*
99 ±13-9
317±25-8*
412 ±69-4*
770±85-8*
Embryonic ectoderm
Mean cell number ± S.E.
Control
Double
221 ±18-5
378 ±46-3*
530 ±30-6
718± 124-7*
76 ±13-7
295±21-7*
369 ±41-2
687 ±103-9*
Extraembryonic ectoderm
Mean cell number ±S.E.
Control
Double
259 ±30-3
411+33-3
474 ±28-6*
676 ±77-9*
2
5
2
4
Number of
embryos
Double Control
97-4 ±0-4
96-6 ±0-8
96-2 ±0-7
98-3 ±0-4
96-6 ±0-7
98O±O-l
96-8 ±0-4
Extraembryonic ectoderm
Mean LI ± S.E.
Double
Control
930±0-6
Embryonic ectoderm
Mean LI ± S.E.
Double
Control
93-0 ±0-8
91-6±0-l
93-4 ±1 •1
90-7 ±2 •5
Endoderm
Mean LI ± S.E.
Control
Double
Table 3. Labelling index of the various tissues in embryos labelled with [3H]thymidine
149±8-6
279 ±11-9
435 ±25-5*
629 ±64-4*
Endoderm
Mean cell number ±S.E.
Double
Control
* Denotes a non significant difference between experimental and control values at P < 0-05 using Student's Mest.
7
5
5
6
Number of
embryos
Double Control
Table 2. Cell number of the embryonic ectoderm, extraembryonic ectoderm and endoderm in double and control embryos
i
g*
>!
2
chan
176
N.E.LEWIS AND J. ROSSANT
Embryonic
ectoderm
50Total
40-
40MI
MI
30-
30-
20-
20I
6 d 0 h
5 d l 6 h
I
6 d 8 h
I
6 d l 6 h
I
7 d 0 h
I
5 d l 6 h
Extraembryonic
ectoderm
I
6 d 0 h
i
6 d 8 h
40Endoderm
T
40-
rr
MI
30-
r
6 d l 6 h
MI
30-
20-
205 d 16 h
6d0h
6d8h
6 d 16 h
5 d 16 h
6d0h
Time p. c.
6d8h
6 d 16h
Time p. c.
Fig. 3. Change in mitotic indices obtained after colcemid treatment of double and
control embryos with time, # = Control embryos; O = double embryos.
Table 4. Cell cycle length {hours) of double and control embryos
derived from mitotic index values
Days p.c.
5dl6h
6d0h
6d8h
6dl6h
Embryonic ectoderm
Control
Double
5-4
4-1
3-7
4-2
6-9
5-4
5-3
4-3
Extraembryonic
ectoderm
Control
Double
7-1
6-7
4-9
5-6
8-0
8-7
6-9
50
Endoderm
Control
Double
12-5
8-0
5-9
6-6
11-8
11-1
9-5
6-9
Mechanism of size regulation in mouse embryo aggregates 177
between 5 days, 16 h and 6 days, 16 h (Fig. 3), peaking at 6 days, 8 h and then
declining. Mitotic activity was highest in the embryonic ectoderm where the M I
after 2 h colcemid treatment reached 53-6 at 6 days, 8 h. Double embryos failed
to show this increase in mitotic activity; large differences in MI were observed
between double and control embryos in all tissues, particularly at 6 days, 0 h
and 6 days, 8 h (Fig. 3). The differences between M i s of double and single
embryos were significant at 6 days, 0 h and 6 days, 8 h (P > 0-05, using Student's
t test) for all tissues except extraembryonic ectoderm. M i s were not significantly
different in any tissue at 5 days, 16 h or 6 days, 16 h.
The mitotic index produced after colcemid treatment was used to estimate
cell cycle length in double and control embryos over the period of size regulation
(Table 4), using the following formula
, ,
,
colcemid treatment time x 100
„
cell cycle length =
-z—rr—; : — — .
percentage of cells in mitosis
This estimate depends on several assumptions (Snow, 1976, 1977):
(1)
(2)
(3)
(4)
(5)
that
that
that
that
that
all cells are dividing,
all cells are dividing at the same rate,
there is no synchrony of cell division,
no cells are entering or leaving the population,
colcemid is not toxic to cells.
Continuous labelling with [ 3 H]thymidine revealed that > 90 % of the cells
(Table 3) were synthesizing D N A and, thus, were presumably mitotically active.
Assumptions (2), (3) and (4) are unlikely to be entirely true (Snow, 1977) but
are common to all methods of estimating cell cycle lengths. Although colcemid
is known to be toxic to cells, we observed no deleterious effects over the 2 h
period used and Copp (1978) has shown that blastocysts can survive exactly the
same colcemid treatment as used here. Thus, we consider that the cell cycle
lengths derived from colcemid-induced M i s represent reasonable estimates on
which to base comparisons between double and single embryos.
Timing of differentiation in double embryos
In all the previous analysis, embryos were grouped according to gestational
age rather than by their stage of development, because it was difficult to recognize distinctive stages of egg cylinder development. However, there are two
morphological events which can be readily scored: proamniotic cavity formation and primitive streak development. Proamniotic cavity formation occurred
around 5 days, 6 h in control embryos, prior to the time of size regulation.
Mesoderm was first observed around 6 days, 16 h, after size regulation was
complete. When these events were scored in double embryos, it became obvious
that proamniotic cavity formation occurred earlier in double than control
embryos. The majority of double embryos had formed the proamnion by 5 days,
5d
5d
6d
6d
6d
16 h
Oh
8h
16 h
8h
Days p.c.
100
100
0(0/5)
14(1/7)
75(6/8)
15(2/13)
65(9/14)
94(17/18)
100
100
75(6/8)
91(10/11)
100(4/4)
100
100
Peicentage embryos forming
proamnion
Half
Control
Double
0
0(0/8)
75(3/4)
0
0
0
0
0(0/15)
86(13/15)
0
2055 ±153
349 ±23
2253 ±313
389 ±46
Mean cell number when majority of
embryos differentiate
Half
Control
Double
0
0
0
316±52
10(1/10)
100(10/10) 1833 ±129
Percentage embryos forming
mesoderm
Half
Control
Double
Table 5. Timing of differentiation in double, control and half embryos
C/3
o
w
w
oo
Mechanism of size regulation in mouse embryo aggregates 179
8 h. The timing of proamnion formation was also studied in half embryos,
produced by destruction of one blastomere of a 2-cell embryo. We have preliminary data (unpublished) that these embryos also size regulate around the
same time as double embryos, although regulation may not be complete until
7 days of development. Proamnion formation was delayed until 6 days, 0 h in
the majority of half embryos (Table 5). The mean cell numbers of double, control
and half embryos were very similar at the time when the majority of embryos
had formed the proamnion. Embryos were also scored for the formation of
mesoderm, which occurred at the end of the size regulation period. Double,
control and half embryos formed mesoderm at the same time and at the same
cell number (Table 5).
DISCUSSION
In this study, double embryos produced by aggregating eight-cell mouse
embryos were shown to remain twice the size of single embryos until at least
5 days, 16 hp.c. Size regulation occurred rapidly over the next 24 h period; there
was no difference in cell number between double and control embryos by 6 days,
16 h p.c. (Table 1,2; Fig. 2). The timing of size regulation found in this study
differs from that observed by Buehr & McLaren (1974). They reported that size
regulation was complete by proamnion formation, which is approximately 24 h
earlier than observed in this study. This discrepancy may result from the different
strains of mice used, but may also reflect the small sample sizes and irregularly
spaced sampling points used by Buehr & McLaren. Sample size in the present
study was never less than four and often much higher, and samples were taken
every 8 h over the complete post-implantation period studied.
Size regulation was found to occur in all tissues of the embryos over roughly
the same time period (Fig. 2). No marked differences in either onset or completion of size regulation were observed. This suggests that there is a single regulatory system controlling size in the early embryo. This differs from the size
regulation seen in later embryos treated with Mitomycin-C where different
tissues regulate for cell loss at different times (Snow & Tarn, 1979).
There are several mechanisms that could account for the size regulation
observed in double embryos (Rossant, 1977):
1. Double embryos might experience enhanced cell death. Cell death is
difficult to assess in the light microscope (Saunders, 1966) but no obvious increased level of necrosis was observed in double embryos. Also, continuous
labelling with [3H]thymidine revealed that > 90 % of cells in double embryos
were synthesizing DNA (Table 3) and were presumably viable.
2. A large non-dividing population of cells might occur in double embryos
around the time of size regulation. Although viable, these cells would be blocked
in some stage of the cell cycle, while other cells of the embryo divided at the
normal rate. Labelling with [3H]thymidine did not reveal any such population
during the initial stages of size regulation (Table 3). Nearly all cells in both
double and single embryos were active in DNA synthesis.
180
N. E. LEWIS AND J. ROSSANT
3. Double embryos might show an increase in cell cycle length compared to
that of controls. Cell cycle lengths calculated from colcemid-induced Mis
(Table 4) provide strong evidence to support this proposed mechanism. Over
the period of size regulation double embryos showed a much lower MI in all
tissues and hence longer cell cycle length than single embryos. Control embryos
showed a proliferative burst, which peaked around 6 days, 8 h, while double
embryos showed a gradual shortening of cell cycle times but no dramatic peak
of mitotic activity (Fig. 3). The proliferative burst observed in control embryos
has been reported previously by Snow (1977) and the cell number and cell cycle
lengths reported here correspond well with his results.
It is difficult to prove conclusively that the difference in MI observed between
double and control embryos is sufficient to account for the entire process of size
regulation. The MI at one time point cannot be used to predict the cell number
at a later point since the cell cycle length changes continuously between the two
time points. However, a rough calculation using differences in cell cycle length
of the order observed serves to illustrate that the differences are of the right
magnitude to account for size regulation. If the cell cycle length of the single
embryos were 6 h and of the double embryos were 8 h, then in 24 h, the double
embryos would undergo three doublings while the single embryos would experience four cell doublings. If double embryos began with 2x cells and controls
with x, both would contain 16x cells after 24 h.
These experiments have clearly established that the length of the cell cycle
can be regulated in the early post-implantation mouse embryo in order to compensate for increased preimplantation cell number. The mechanism by which
cell cycle length is altered remains to be elucidated but it is interesting to note
that size regulation occurs when the normal embryo is undergoing a dramatic
increase in mitotic activity. This suggests that this may be an important control
point in development at which cell division rate can be increased or decreased
to compensate for any deviations from normal cell number. It is not possible to
determine from the present study whether size regulation is intrinsic to the
embryo or whether the maternal environment plays a role. Study of size regulation of double embryos in vitro will be necessary to answer this question.
The results of this study also provide some information about the timing of
differentiation in the early postimplantation embryo. The first differentiative
event in postimplantation development which can be readily scored morphologically is proamnion formation. This event occurred before size regulation
but the time when a majority of embryos had formed a proamnion was found
to differ, with most double embryos forming the cavity before controls and most
controls before halves. However, it is interesting to note that the mean cell
numbers of double, control and half embryos were almost identical at the time
when a majority of each of them had formed the proamnion (Table 5). This
suggests that the timing of differentiation in the postimplantation embryo may
depend on the absolute cell number. This contrasts with differentiation in the
Mechanism of size regulation in mouse embryo aggregates 181
preimplantation embryo where blastocoel formation has been shown to occur
independently of total cell number. This event is believed to be timed by the
number of nuclear divisions undergone since fertilization or by some other
intrinsic cytoplasmic clock set in motion at fertilization (Smith & McLaren,
1977; Johnson, 1981). Experiments investigating differentiative events at a
biochemical rather than a morphological levelwill clearly have to be undertaken
in order to verify that differentiation in the postimplantation embryo utilizes
a different timing mechanism from the preimplantation embryo.
We should like to thank Dr V. E. Papaioannou for useful discussion. The work was
supported by the Canadian Natural Sciences and Engineering Research Council.
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(Received 25 March 1982, revised 18 May 1982)