J. Embryol. exp. Morph. 94, 139-148 (1986)
139
Printed in Great Britain © The Company of Biologists Limited 1986
Size regulation in the mouse embryo
I. The development of quadruple aggregates
G. F. RANDS
Sir William Dunn School of Pathology, University of Oxford, South Parks Road,
Oxford, OX1 3RE, UK
SUMMARY
The development of mouse embryos formed by the aggregation of four 8-cell-stage eggs was
analysed in comparison with control single embryos. The analysis revealed that:
(1) Quadruple aggregates undergo size regulation over several days, starting before implantation and being completed by 6-5 days post coitum.
(2) The attainment of recognizable postimplantation morphological stages is independent of
size.
(3) Regulation is not brought about by disproportionate alterations in the size of the internal
cavities.
(4) Regulation in both inner cell mass (ICM) and trophectoderm derivatives is completed
between 5-5 and 6-5 days post coitum.
(5) Despite the abnormal proportions of ICM and trophectoderm in quadruple blastocysts,
the proportions of the tissues derived from them are already normal by 5-5 days.
The possibility that down regulation in size of aggregate embryos occurs as a consequence of
limited nutrient supply is discussed.
INTRODUCTION
Chimaeras produced by the aggregation of early embryos have been widely used
in experimental investigations of mammalian embryology and developmental
genetics (McLaren, 1976; Le Douarin & McLaren, 1984). The fact that aggregates
of two (Tarkowski, 1961; Mintz, 1962,1965), three (Markert & Petters, 1978), four
(Petters & Markert, 1980) or even up to nine (Petters & Mettus, 1984) preimplantation mouse embryos can result in offspring which are apparently entirely
normal in size and proportions demonstrates the remarkable regulatory powers of
the mammalian embryo.
Some aspects of this phenomenon of size regulation have been investigated in
double embryo aggregates (Buehr & McLaren, 1974; Lewis & Rossant, 1982). But
little information is available concerning the crucial relationship between size
regulation and the progression of developmental events. Nor has the regulation of
larger-sized aggregates been examined, despite the fact that hexaparental and
octoparental aggregation chimaeras are now being used in genetic and developmental studies (Markert & Petters, 1978; Petters & Markert, 1980). Moreover, it
has recently been shown that at the blastocyst stage octoparental chimaeras
Key words: size regulation, mouse embryo, aggregation chimaera, growth.
140
G. F. RANDS
(quadruple aggregates) have a disproportionately large inner cell mass (ICM) and
disproportionately small trophectoderm when compared with standard embryos
(Rands, 1985).
The present study presents an analysis of the further development and regulation of such quadruple (quad) aggregates, with particular reference to the timing
of morphogenetic and differentiative events.
MATERIALS AND METHODS
Production of embryos
Embryos were obtained from natural matings of random-bred CFLP mice (Anglia Laboratory
Animals Limited). PB1 medium (Whittingham & Wales, 1969) containing glucose (lgl" 1 ) in
place of lactate and foetal calf serum (10 % v/v) in place of bovine serum albumin (Gardner,
1982) was used for recovery and transfer of embryos.
8-cell embryos were flushed from females at 13.00-15.00 h on the third day of pregnancy and
their zonae removed by brief exposure to acidic Tyrodes solution (Nicolson, Yanagimachi &
Yanagimachi, 1975). Embryos were cultured singly (controls) or in aggregates of four, as
described by Rands (1985). After incubation for 24 h all healthy and well-integrated morulae
were transferred, generally in groups of six per horn, to the uteri of females on the third day of
their pseudopregnancy (McLaren & Michie, 1956; McLaren, 1969); the females had been mated
with vasectomized males and were anaesthetized using Avertin (Winthrop, UK). Aggregate and
control embryos were placed in opposite horns of the same female (Buehr & McLaren, 1974).
The overall implantation rate for females that became pregnant was 79 % for control embryos
and 64 % for quads (Yates' chi-squared value = 0-72, P > 0-05). Embryo age at postimplantation
stages, quoted as days post coitum (p.c), refers to the age of pseudopregnancy in the recipient
female.
Histology
At 5-5, 6-5 and 7-5 days p.c., uterine horns containing implantation sites were recovered and
fixed overnight in Bouin's fluid. They were then dehydrated, embedded in paraffin wax (melting
point 56 °C), serially sectioned at 7 [im, and the sections stained with haemalum and eosin.
Volume estimations
Serial sections of entire embryos or parts of embryos were drawn out at a fixed magnification
of x200 using a Zeiss drawing tube. For the purpose of standard and repeatable drawing, the
'entire embryo' was taken to include the internal cavities and the coherent core cells of the
ectoplacental cone but to exclude Reichert's membrane (Fig. 1).
The area of each embryonic section was measured from the drawings by means of a semiautomatic image analysis apparatus, in which a digitizing pad (Summergraphics Bitpad) with a
tracing stylus feeding a microcomputer (SWTPC 6800) was used to outline the sections. An
estimate of the volume of the whole embryo or component part was then obtained by summing
the section areas and multiplying by the section thickness.
Volume estimations of single and quad embryos were obtained for:
(a) the entire embryo;
(b) the proamniotic (at 5-5 and 6-5 days) or amniotic (at 7-5 days) cavity;
(c) the postimplantation tissues which are derived from the trophectoderm of the blastocyst
(see Fig. 1);
(d) the postimplantation tissues which are derived from the ICM of the blastocyst (see Fig. 1).
Morphological assessment of developmental stage
The developmental stage of postimplantation embryos was assessed by reference to a set of
criteria (Table 1) drawn up from the work of Jolly & Ferester-Tadie (1936), Snell & Stevens
Size regulation in quadruple aggregates
Ectoplacental cone "
(coherent core cells)
Extraembryonic
ectoderm
141
Trophectoderm
derivatives
Visceral endoderm
Mesoderm
ICM
derivatives
Embryonic ectoderm
Proamniotic cavity
Fig. 1. Diagram of the egg cylinder.
(1966) and Buehr & McLaren (1974). The plane of section was approximately frontal
(longitudinal) to the embryo, though it may be noted that this becomes transverse with respect
to the midtrunk region after the egg-cylinder stage.
Statistical analysis
Measurements from quad and control embryos were compared using Student's t-test (Bailey,
1959) or, if the sample variances differed significantly, using Satterthwaite's variation
(Satterthwaite, 1946).
Table 1. Morphological staging criteria for the mouse at 4-5-8-5 days post coitum
Stage*
1 (3)
2 (4)
3 (5)
4
5 (6)
6
7
8 (7)
9
10 (8)
11
12
13
14
Egg cylinder partly formed, thickening of ectoderm but no obvious extraembryonic
ectoderm
Egg cylinder fully formed, embryonic ectoderm distinct from extraembryonic
Proamniotic cavity beginning to form
First appearance of ectoplacental cone
Cavity extending into extraembryonic ectoderm
Visceral endoderm over (distal) embryonic ectoderm squamous compared to columnar
visceral endoderm over extraembryonic ectoderm
Appearance of (lateral) mesoderm as clear migrating layert
Cavities in mesoderm of amniotic folds: beginning of exocoelom
Closure of amnion
Allantois seen (from edge of amnion, at posterior)
Head process seen at ventral edge of egg cylinder
Ectoderm forms definite V-shaped trough (beginning of neural groove) in anterior half
of embryonic region
Ectoplacental cavity eliminated by chorion pushing up against ectoplacental cone
Allantois contacts chorion
* Numbers in parentheses indicate the stages used by Buehr & McLaren (1974).
t Because of the frontal plane of sectioning, one is mainly looking at the lateral amniotic folds
and it is difficult to distinguish the posterior (and anterior) folds. Thus thefirstappearance of the
mesoderm at the primitive streak is not easily seen.
142
G. F. RANDS
RESULTS
Overall course of size regulation
The results of size measurements - cell number at preimplantation stages (data
from Rands, 1985) and egg cylinder volume at postimplantation stages - show that
quad embryos cease to be significantly different from controls between 5-5 and 6-5
days p.c. (Table 2). However, the ratio of the mean sizes of quad and control
embryos at each age (Table 2) falls steadily over several days, t-tests show that
although the ratio at the morula stage is not statistically different from 4-0, by the
blastocyst stage it has already dropped significantly below this level. The ratio at
5-5 days is even lower than that at the blastocyst stage, but the difference between
these two is not statistically significant (t-test: P > 0-05) - thus there is no evidence
for regulation occurring actually over the time of implantation.
Developmental staging of postimplantation embryos
Table 3 shows the developmental stages reached by the postimplantation embryos examined. The criteria used are seen to distinguish the three age groups but
do not reveal any clear differences between quad and control embryos of the same
age.
The volume estimates for the embryos may also be grouped according to the
developmental stage reached (Table 4: only at stages 2, 3, 6 and 11 are there
embryos in both experimental and control classes). It is seen that quad and control
embryos are significantly different in size at stages 2 and 3, but are not so by stages
6 and 11.
Table 5 addresses specifically the question of the timing of differentiation, as
exemplified by two clearly recognizable features: the formation of the proamniotic
cavity (stage 3) and the appearance of the mesoderm (stage 7). The data indicate
that there is no difference between quad and control embryos in the timing of
either of these events. However, since proamnion formation occurs before size
regulation is complete, quad embryos are still substantially larger at this time.
Proportions of component parts of the egg cylinder
The proportion of the total egg cylinder volume occupied by the proamniotic (at
5-5 and 6-5 days) or amniotic (at 7-5 days) cavity does not differ significantly
between quad and control embryos (Table 6). Table 6 also shows that the volumes
of both ICM- and trophectoderm-derived tissues are significantly greater in quads
than controls at 5-5 days but are not so at 6-5 days. However, the proportion of the
total tissue mass which is occupied by ICM-derived (and hence also that occupied
by trophectoderm-derived) tissue is the same in quads and controls at both 5-5 and
6-5 days (Table 6).
19-5 ±2-5 (9)
246-9 ± 39-4 (12)
3598-6 ±396-0 (4)
21-9 ±1-1 (17)
62-8 ±3-4 (11)
Number of
embryos
9
15
12
7
4
2
See Table 1 for key to stage numbers.
Age Control
(days) or quad
5-5
control
quad
6-5
control
quad
7-5
control
quad
51-6 ±7-1 (15)
346-9 ± 56-0 (7)
3589-2 ± 1174-2 (2)
32
94-5 ±2-0 (10)
207-8 ± 4-6 (8)
NS
NS
33
53
67
40
—
14
100
86
10
% of embryos at each developmental stage*
100
100
11
Table 3. Developmental staging of postimplantation quad and control embryos
* t-test compares quad and control.
tt-test compares observed ratio with the expected value of 4-0.
tData from Rands, 1985.
§ Volumes are expressed as //m 3 xl0 4 .
** Significant difference (P<0-05).
NS, non-significant difference.
Egg cylinder volume: 5-5 days§
6-5 days
7-5 days
Cell number: at aggregation
morula$
blastocyst$
12
2-6
1-4
1-0
4-0
4-3
3-3
Table 2. The sizes of quad and control embryos at preimplantation and early postimplantation stages
Mean size ± S.E.M. (no. of embryos)
Mean size
ratio
Control
t-test*
Quad
13
**
**
**
NS
**
14
t-testf
I
oa
144
G. F. RANDS
Table 4. The volume of postimplantation quad and control embryos as a function of
their developmental stage
Mean volume* ± S.E.M. (number of embryos)
Stage
2
3
6
11
Control
11-6 ±4-0 (3)
23-3 ± 1-7 (6)
246-9 ± 39-4 (12)
3598-6 ± 396-0 (4)
Quad
35•7± 3-6(8)**
59•4± 5-3 (6)**
381•8± 52-0 (6) NS
3589•2± 1174-2 (2) NS
Mean volume
ratio
3-1
2-5
1-5
1-0
* Volumes expressed as jum3xl04.
t See Table 1 for key to stage numbers.
** Significantly different from control (P< 0-05) by t-test.
NS, not significantly different from control.
Table 5. Differentiation in postimplantation quad and control embryos
% of embryos which have formed proamnion: at 5-5 days
at 6-5 days
Volume of largest embryo which has not formed proamnion"
Volume of smallest embryo which has formed proamnion*
% of embryos which have formed mesoderm: at 6-5 days
at 7-5 days
Control
Quad
67 (6/9)
100 (12/12)
19-0
17-7
0(0/12)
100 (4/4)
47 (7/15)
100 (7/7)
50-1
43-6
0(0/7)
100 (2/2)
* Volume expressed as |Wm3xl04.
DISCUSSION
Quadruple mouse embryos, produced by the aggregation of four morulae,
appear to undergo size regulation over several days. By the blastocyst stage the
ratio of quad: single embryos sizes has already dropped significantly below the 4:1
ratio which was produced at the time of aggregation and maintained at the morula
stage. Thus there is evidence that some regulation occurs before implantation in
quad embryos, whereas double embryos show no sign of regulation until after
implantation (Buehr & McLaren, 1974; Lewis & Rossant, 1982). By 5-5 days post
coitum quad embryos are little more than twice the size of control embryos. Over
the next 24 h they undergo the final stages of regulation in parallel with double
embryos (Lewis & Rossant, 1982) produced using a very similar experimental
procedure. Size regulation in quads is complete by 6-5 days p . c , at which time
the proamniotic cavity has extended into the extraembryonic ectoderm but the
primitive streak has not yet appeared.
Postimplantation quad embryos are at about the same developmental stage as
control embryos of the same chronological age, both before and after regulation.
This has also been noted for double embryos by Buehr & McLaren (1974), but
conflicts with data of Lewis & Rossant (1982). In particular, the present results
0-7 ±0-2 (7) NS
32-5 + 6-1(9)**
26-8 ±4-3 (9)**
54-2 ± 1 0 (9) NS
0-5 ±0-1 (6)t
12-1 ±1-2 (8)
9-1 ± 1-0 (8)
55-7 ± 2-8 (8)
Quad
* Proamniotic cavity at 5-5 and 6-5 days, amniotic cavity at 7-5 days.
t Numbers in parentheses represent sample sizes.
** Significantly different from control (P< 0-05) by t-test.
NS, not significantly different from control.
Cavity volume as mean %
of overall embryo vol. ± S.E.M.*
Tissue vol. (/mi 3 xl0 4 ) ± S.E.M.
(a) ICM-derived tissues
(b) trophectoderm-derived tissues
ICM tissue as % of total vol. ± S.E.M.
Control
5-5 days
147-8 ±21-9 (8)
124-5 ± 19-1 (8)
54-0 ±2-1(8)
3-4 ±0-5 (12)
Control
Quad
203-8 ± 43-8 (5) NS
148-9 ± 30-0 (5) NS
58-2 ± 3-2 (5) NS
3-3 ± 0-7 (7) NS
6-5 days
7-5 days
—
—
—
14-5 ± 2-8 (4)
Control
Table 6. Cavity and tissue volumes of postimplantation quad and control embryos
13-7 (1) NS
Quad
1
i
3
CX3
Op
146
G. F. RANDS
indicate that quad embryos form the proamniotic cavity at about the same
chronological age as controls but are significantly larger at this time; Lewis &
Rossant (1982), on the other hand, showed that proamnion formation in doubles
occurred earlier than in controls and at a similar cell number in both. The
explanation of this apparent discrepancy between double- and quadruple-sized
embryos is difficult to see, and the two sets of results have crucially different
implications for theories about the mechanism of timing of differentiation in
postimplantation embryos. The analysis of quad embryos shows that proamnion
formation is independent of size - as is the formation of the blastocoel at the
preimplantation stage (e.g. Smith & McLaren, 1977). By contrast, the data of
Lewis & Rossant (1982) indicate that proamnion formation is dependent on the
absolute cell number and therefore that a different sort of mechanism must be
postulated for postimplantation embryos. The resolution of this issue is clearly
important.
An analysis of the volumes of component parts of the egg cylinder (see Fig. 1)
reveals, firstly, that the proportion of the total volume occupied by the internal
cavities is equal in quad and control embryos of the same age. Thus the pattern of
size changes seen during regulation is not brought about by disproportionate
alterations in cavity volume. Secondly, the analysis reveals that the volumes of
tissues derived from both the ICM and the trophectoderm in quads are significantly different from those in single embryos at 5-5 daysp.c.; at 6-5 days, by which
time overall size regulation is complete, they are no longer significantly different.
This indicates that regulation is occurring in both lineages over the same time
period.
But let us now consider the proportions of ICM- and trophectoderm-derived
tissues in regulating and standard embryos. At 6-5 daysp.c. the proportions in
quad embryos are found to be the same as those in controls. Therefore regulation
has occurred in this respect also, when compared with the significant differences
seen between ICM and trophectoderm proportions in quad and single embryos
at the blastocyst stage (Rands, 1985). However, when the quad embryos are
examined at 5-5 days, before overall size regulation is complete (although some
regulation has occurred), the proportions of ICM- and trophectoderm-derived
tissues are already normal. This result is unexpected, particularly in the light of the
wide discrepancy seen at the blastocyst stage.
In view of the fact that double and quad embryos both complete their regulation
at the same chronological age, it may be postulated that there is a single 'window'
in embryonic development during which down regulation of size must take place.
The period around implantation is one of extremely rapid growth and morphological change. The formation of the 7-5-day embryo from the blastocyst requires
more than a 500-fold increase in tissue volume (Snow, 1976). In a situation where
the embryo is much larger than normal, it seems likely that the dominant and
limiting factor regarding growth may be the availability of metabolites or growthpromoting substances, possibly supplied to the embryo via its external surface. For
a given shape, the surface area is relatively reduced as the volume increases (see
Size regulation in quadruple aggregates
147
Rands, 1985) and therefore such an effect might well be expected to be considerably more serious on quad-sized embryos than doubles. This is supported by
the observation that regulation commences earlier in quad embryos than in
doubles. Moreover, one might also expect the initial regulatory effect to be more
severe on the (internal) ICM than on the (external) trophectoderm - which may
account for the observation that by 5-5 days the proportion of ICM tissue has
already dropped to the normal level.
Since quad embryos are not developmentally advanced when compared with
control embryos of the same age, it appears that increasing size cannot speed up
the rate of morphogenesis, i.e. there has been an unhitching of morphogenetic
stage from size. One conclusion that may be drawn from this is that regulation
must be due to an alteration in the rate of growth rather than the rate of
morphogenesis. A simple comparison of the numbers of mitotic and dead cells in
the ICM- and trophectoderm-derived tissues of 5-5- and 6-5-day embryos did not
detect a significant difference between quads and controls (G. F. Rands,
unpublished observations). However, where colcemid has been used to amplify
greatly the observed mitotic indices, evidence has been obtained that regulation in
double aggregates is brought about by a longer cell cycle time in these embryos
than in standard ones (Lewis & Rossant, 1982).
The behaviour of chimaeric aggregates is only one aspect of the capacity of the
mouse embryo for size regulation. An analysis of regulation in half-sized embryos
will be presented in the second paper (G. F. Rands, in preparation) and will
include a comparison of the features of up and down regulation.
I would like to thank Mr D. G. Papworth of the MRC Radiobiology Unit at Harwell for
statistical advice, and Dr S. Bradbury and Miss A. Stanmore of the Department of Human
Anatomy at Oxford for kindly allowing me to use the image analysis apparatus. I also thank Dr
J. D. West, Professor R. L. Gardner, Dr R. Beddington and Dr M. R. W. Rands for all their
advice and encouragement. This work was supported by a Medical Research Council
Studentship.
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