/. Embryol. exp. Morph. 98, 209-217 (1986)
209
Printed in Great Britain © The Company of Biologists Limited 1986
Size regulation in the mouse embryo
II. The development of half embryos
G. F. RANDS
Sir William Dunn School of Pathology, University of Oxford, South Parks Road,
Oxford OX1 3RE, UK
SUMMARY
The study describes an analysis of the development of mouse embryos halved at the 2-cell
stage by the destruction of one blastomere, in comparison with control embryos of parallel
derivation, at 2-5-13-5 days post coitum. The results showed that: (1) half embryos achieve size
regulation some time between 7-5 and 10-5 days; (2) there is an indication that by 13-5 days half
embryos may have again dropped back significantly in size relative to controls; (3) preregulation
half embryos are slightly retarded developmental, but this does not wholly account for their
smaller size: morphogenesis is not size-dependent; (4) early postimplantation half embryos
contain a significantly decreased proportion of inner cell mass derivatives and increased proportion of trophectoderm derivatives when compared with controls.
A comparison is also made between the up-regulation of half embryos and the downregulation of aggregate embryos, and it is suggested that size regulation may occur by delaying a
change in the normal growth rate.
INTRODUCTION
Cleavage-stage embryos of a variety of mammals in which one or more
blastomeres had been destroyed or separated have been shown to be capable of
further development (Nicholas & Hall, 1942; Tarkowski & Wroblewska, 1967;
Moore, Adams & Rowson, 1968; Willadsen, 1980; Willadsen, Lehn-Jensen,
Fehilly & Newcomb, 1981; Allen & Pashen, 1984). Continued development was
demonstrated both in vitro, by the formation of blastocysts, and in utero, after
transfer to pseudopregnant recipients. In the mouse, single blastomeres from
2-cell embryos can give rise to normal postimplantation conceptuses and live
young (Tarkowski, 1959a,b; Hoppe & Whitten, 1972; Tsunoda & McLaren, 1983)
despite the fact that the blastocyst formed from such a half embryo contains
significantly disturbed proportions of inner cell mass (ICM) and trophectoderm
when compared with standard embryos (Rands, 1985).
The present study investigates the course of postimplantation growth and
morphogenesis in half embryos and extends a previous investigation (Tarkowski,
19596) in the following ways: (a) the use of control embryos which were shamoperated and transferred to pseudopregnant recipients in parallel with the experimental group; (b) the assessment of developmental stage by reference to standard
criteria; (c) the application of statistical tests to the data obtained.
Key words: size regulation, mouse, half embryo, morphogenesis, growth.
210
G. F. RANDS
A comparison is also made between this capacity of the mouse embryo for
up-regulation after a reduction in preimplantation size and its capacity for downregulation after the aggregation of preimplantation embryos (Buehr & McLaren,
1974; Lewis & Rossant, 1982; Rands, 1986).
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 ( l g P 1 ) in
place of lactate and foetal calf serum (10 % v/v) in place of bovine serum albumin (Gardner,
1982) was used for recovery, manipulation and transfer of embryos.
2-cell embryos were flushed from the oviducts of females at 13.00-15.00 h on the second day
of pregnancy. As described previously (Rands, 1985), half embryos were produced by the
mechanical lysis of one blastomere; control embryos were treated in exactly the same way as the
experimental group except that the zona pellucida alone was penetrated. Half and control
embryos were then transferred to opposite oviducts of females on the first day of pseudopregnancy, generally six embryos per oviduct.
The overall implantation rate for females that became pregnant was 88 % for controls and
73 % for half embryos (Yates' chi-squared value = 0-46, P > 0-05). Embryonic age after transfer
was recorded as days post coitum (p.c.) of recipient rather than donor females (Marsk, 1977).
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 7jUm and the sections stained with haemalum and eosin. The
plane of section was approximately frontal (longitudinal) to the embryo.
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 coherent core cells of the
ectoplacental cone but to exclude Reichert's membrane (see Rands, 1986, fig. 1). The area of
each embryonic section was measured from the drawings by means of a semi-automatic image
analysis apparatus (Rands, 1986). 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 control and half embryos were obtained for: (a) the entire embryo at
5-5, 6-5 and 7-5 days; (b) the proamniotic (at 5-5 and 6-5 days) or amniotic (at 7-5 days) cavity;
(c) the tissues at 6-5 days that are derived from the trophectoderm of the blastocyst (extraembryonic ectoderm and ectoplacental cone); (d) the tissues at 6-5 days that are derived from
the ICM of the blastocyst (embryonic ectoderm, visceral endoderm and mesoderm).
Morphological assessment of developmental stage
The developmental stage of postimplantation embryos at 4-5-8-5 daysp.c. was assessed from
histological sections by reference to the criteria listed in Table 1.
Dry weight measurements
Conceptuses were recovered at 10-5, 12-5 and 13-5 days p.c. and separated into embryonic
and placental fractions (the extraembryonic membranes were discarded). Dry weights were
measured to the nearest 0-1 mg after 3 h at 150°C.
Size regulation in half embryos
211
Table 1. Morphological staging criteria for the mouse at 4-5-8-5 days post coitum
(from Rands, 1986)
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 layerf
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 end 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 brackets 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.
Statistical analysis
Measurements from half and control embryos were compared using Student's Mest
(Bailey, 1959) or, if the sample variances differed significantly, using Satterthwaite's variation
(Satterthwaite, 1946).
RESULTS
Overall course of size regulation
Table 2 shows the results of size estimations (cell number, embryo volume or
dry weight) for half and control embryos at 2-5-13-5 days p. c. Up to and including
7-5 days, Mests reveal first that the sizes of half embryos are significantly different
from controls, and second that the ratios of half: control sizes are not significantly
different from the initial ratio of 0-5:1. Sampling at later stages of gestation shows
a different situation. By 10-5 days regulation has occurred: at 10-5 and 12-5 days
there is no significant difference between the sizes of half and control conceptuses,
in either embryonic or placental fraction.
At 13-5 days it may be seen that half conceptuses are again significantly smaller
than controls when compared directly by Mests (Table 2). However, when the
ratios at 10-5 and 12-5 days are compared with those at 13-5 days a statistically
significant difference is not demonstrable (P>0-05).
control
half
control
half
control
half
5-5
17
10
11
13
5
4
Number of
embryos
6
30
—
—
20
—
—
* See Table 1 for key to stage numbers.
7-5
6-5
Control
or half
Age
(days)
s
S
S
S
S
S
NS
NS
NS
NS
S
/-test*
0-50
0-44
0-50
0-51
0-35
0-33
1-01
0-88
0-79
0-77
0-72
0-77
Mean size
ratio
—
88
50
—
6
9
100
_
_
91
—
_
—
—
—
_
8
_
—
25
_
—
25
10
_
% of embryos at each developmental stage*
20
25
11
_
Table 3. Developmental staging of early postimplantation half and control embryos
* /-test compares half and control groups.
f/-test compares observed ratio with the expected value of 0-5.
t Data from Rands, 1985.
§ Volumes expressed as jum 3 xl0~ 4 .
1f Dry weights expressed as mgx 10.
S Significant difference (P<0-05).
NS Nonsignificant difference.
Cell number^: 1-5 days
2-5 days
3-5 days
Egg cylinder volume§: 5-5 days
6-5 days
7-5 days
Dry weight^: 10-5 day embryo
10-5 day placenta
12-5 day embryo
12-5 day placenta
13-5 day embryo
13-5 day placenta
Mean size ± S.E .M. (no. of embryos)
Half
Control
1
2
9-2 ±0-6 (5)
20-7 ±2-1 (11)
51-6 ±2-2 (8)
25-5 ± 2-9 (7)
46-3 ±3-7 (17)
23-5 ±3-4 (10)
548-7 ±21-8 (11)
189-7 ± 10-7 (13)
8141-7 ±876-4 (5)
2675-9 ±756-6 (4)
10-0 ±1-0 (12)
10-1 ±1-2 (7)
8-8 ±1-0 (12)
7-7 ±0-6 (7)
75-6 ±4-7 (7)
60-1 ±4-8 (9)
54-1 ±2-9 (7)
41-7 ±5-6 (9)
133-0 ±5-1 (12)
95-4 ±3-7 (9)
90-7 ±3-5 (12)
69-7 ±3-9 (9)
Table 2. The sizes of half and control embryos
80
25
_
12
s
s
s
s
s
NS
NS
NS
NS
NS
S
r-testf
_
13
_
14
p
to
h
Size regulation in half embryos
213
Table 4. The volume of postimplantation half and control embryos as a function of
their developmental stage
Stagef
2
3
6
11
12
Mean volume* ± S.E.M. (no. of embryos)
J
1
'
Control
Half
35-0 (1)
45-1 ±3-6 (15)
595-8(1)
6531-8(1)
8544-1 ± 1005-0 (4)
17-6 ±0-6 (3)
31-0 ±4-6 (5)
189-7 ± 10-7 (13)
3518-2(1)
4283-7(1)
x,
,
Mean volume
ratio
0^50
0-69
0-32
0-54
0-50
* Volumes expressed as jum3xl0~4.
t See Table 1 for key to stage numbers.
Developmental staging of early postimplantation embryos
If the preregulation postimplantation embryos (5-5-7-5 days/?.c.) are analysed
for developmental stage, the results (Table 3) show that half embryos tend to lag
slightly behind control embryos of the same age. This is particularly striking at 6-5
days, when all but one of the control embryos showed clear development of the
lateral mesoderm (stage 7) whereas none of the half embryos did so.
Table 4 shows the volume estimations at 5-5-7-5 days grouped by the developmental stage of the embryos (only at stages 2, 3, 6, 11 and 12 are there representatives from both experimental and control classes). Again the half: control ratio is
around 0-5:1. Thus the fact that half embryos are apparently somewhat less
developmentally advanced than their control contemporaries does not wholly
account for their smaller size. Indeed, the average volume of a half embryo at
stage 3 (or stage 12) is less than the corresponding volume of a control embryo at
stage 2 (or stage 11).
Proportions of component parts of the egg cylinder
Table 5 shows a comparison of the proportion of the total volume occupied
by the proamniotic or amniotic cavity in 5-5-7-5 day embryos. This reveals no
statistically significant difference between half and control embryos.
At 6-5 days half embryos are still considerably smaller than controls and it
is therefore not surprising that the volumes of both ICM and trophectoderm
derivatives are significantly different in the two groups of embryos (Table 5).
However, in Table 5 the half embryos are also seen to maintain a significantly
smaller proportion of ICM derivatives (and larger proportion of trophectoderm
derivatives) than control embryos.
DISCUSSION
The present study demonstrates that mouse embryos derived from one blastomere at the 2-cell stage, when compared with control embryos of exactly parallel
derivation, achieve regulation of their size some time between 7-5 and 10-5 days
post coitum (8—11th day). This substantiates the indication from the work of
214
G. F. RANDS
Table 5. Component volumes of half and control embryos at early postimplantation
stages
Control
Cavity volume* as mean % of overall
volume ±S.E.M.: 5-5 days
6-5 days
7-5 days
Tissue volume^ at 6-5 days ± S.E.M.
(a) ICM-derived tissues
(b) trophectoderm-derived tissues
ICM tissue as % of total volume at
6-5 days ± S.E.M.
0-6±0-l(17)t
3-2 ±0-3 (11)
13-8 ± 2-9 (5)
326-3 ± 13-6 (8)
197-8 ± 9-2 (8)
62-3 ± 1-0 (8)
Half
0-5 ±0-1 (5) NS
2-5 ± 0-2 (13) NS
9-0 ±2-3 (4) NS
94-0 ± 7-4 (8) S
87-3 ± 6-9 (8) S
51-9 ± 0-8 (8) S
* Proamniotic cavity at 5-5 and 6-5 days, amniotic cavity at 7-5 days.
f Numbers in parentheses represent sample sizes.
$ Volumes expressed as jum3xl0~4.
S Significantly different from control (P< 0-05) by Mest.
NS Not significantly different from control.
Tarkowski (1959b) that regulation is completed around the 10-11th day, and
contrasts with a statement by Lewis & Rossant (1982) that these authors have
"preliminary data (unpublished) that [half embryos] size regulate around the same
time as double embryos", i.e. between 5-7 and 6-7 daysp.c.
In contrast to Tarkowski (19596), sampling at 13-5 days in this study suggests
that by the 14th day half embryos may have again dropped back significantly in size
relative to controls (although the statistical tests are not conclusive - see Results).
Tsunoda & McLaren (1983) report that at 18 days the average weight of foetuses
from a large sample of half embryos (produced at the 8- to 16-cell stage and
transferred to the oviduct or uterus after one or two days in culture) is significantly
lower than that of controls. Thus it seems that half embryos, after undergoing size
regulation at mid-gestation, may indeed subsequently fall back.
There may be a parallel to this situation in the development of XO mice.
Burgoyne, Tam & Evans (1983) compared the growth and development of XO and
XX mice between 7 and 19 days of gestation and found that XO egg cylinders at
7-25 days were only half the volume of XX controls. Between 10-5 and 12-5 days
the mean weight of XO foetuses caught up with that of their XX littermates, but
then fell behind again in the second half of gestation so that XO mice were
underweight at birth (Burgoyne, Evans & Holland, 1983).
As shown in this study, preregulation half embryos exhibit slight developmental
retardation compared to controls of the same age. This fact might be taken to
indicate that there is some linkage of morphogenetic stage to size. However, even
if half and control embryos are matched for stage, rather than age, it is clear that
the successive stages of morphogenesis during the first half of gestation occur at a
smaller size in half embryos than is the case in control embryos. In other words,
although half embryos do lag behind in terms of developmental stage, this lag is
less than would be expected if development were entirely size-dependent. Thus
Size regulation in half embryos
215
the normal relationship between morphogenesis and growth has been disturbed
and it can be seen that to some degree each can be independent of the other.
Lewis & Rossant (1982) present some data that indicate that proamniotic cavity
formation is delayed in half embryos and does not occur until they reach the size at
which the cavity forms in controls. However, it is difficult to assess this evidence,
which appears to conflict with the present results, since details of the experimental
procedure are not given; the exact derivation of the control embryos used is
particularly important.
An analysis of the volumes of component parts of the egg cylinder in half
and control embryos reveals that the size discrepancy before regulation in half
embryos is not attributable to a disproportionate difference in the size of the
internal cavities. The analysis also reveals that the ratio of ICM-derived tissue
(embryonic ectoderm, visceral endoderm and mesoderm) to trophectodermderived tissue (extraembryonic ectoderm and ectoplacental cone) in half embryos
at the egg cylinder stage (6-5 days) is significantly smaller than in controls. This
is consistent with results at the preimplantation stage, which show that half
blastocysts have a significantly smaller proportion of ICM and greater proportion
of trophectoderm than control blastocysts (Rands, 1985). It is therefore clearly
possible to sustain postimplantation development despite an abnormal relationship between component parts of the embryo.
A somewhat similar, though more extreme, situation arises after the administration of the drug mitomycin C (MMC) to mouse embryos in utero (Snow & Tarn,
1979). MMC causes massive random cell death when administered at around 7-5
days p. c., yet at least some of the embryos develop into normal functional animals.
The pattern of cell allocation in half embryos revealed by the results at 6-5 days
poses the question of what happens at later stages of development. It would
undoubtedly be of interest to continue to monitor the proportions of tissues in half
embryos up until, and perhaps beyond, the time of their overall size regulation.
Clearly there is a great deal more underlying flexibility in embryogenesis than
is normally revealed in undisturbed development. The embryo has evolved a
considerable regulative capacity which allows the complex developing system
to withstand a range of perturbations. If we make a comparison between upregulation as exemplified by the development of half embryos described above,
and down-regulation as seen in the development of quadruple aggregate embryos
(Rands, 1986), two important points are apparent. First, the attainment of
recognizable early postimplantation stages is not size-dependent: preregulation
quadruple embryos reach them at a larger size and half embryos at a smaller size
than controls.
Second, the outstanding difference between down- and up-regulation is their
timing during pregnancy: the former occurring at around 6 daysp.c. and the latter
at 8-11 days. McLaren (1976, fig. 2) has constructed a composite graph of the
tissue volume and dry weight (drawn on a logarithmic scale) of normal mouse
embryos from fertilization to birth. The form of the curve is an S-shape and it is
notable that the changes in slope occur at about 6 and 10 days. This suggests that
216
G. F. RANDS
regulation may occur at times when the undisturbed embryo is undergoing a
change in growth rate. Thus down-regulation coincides with the beginning, and
up-regulation possibly with the end, of a spurt in growth. In other words, size
regulation may occur by delaying a change in the normal growth rate, rather than by
introducing an entirely novel event into the growth pattern.
The different times at which upward and downward changes occur may reflect
the changing conditions of maternal/foetal relationships (Snow, Tarn & McLaren,
1981). At the early egg cylinder stages the growth of large embryos may be
restricted by reliance on the supply of nutrients by diffusion (Rands, 1986),
thereby accounting for down-regulation. It has been suggested that at around the
middle of gestation the development of efficient exchange via the chorioallantoic
placenta may allow smaller embryos to up-regulate (Tarkowski, 1959£>). However,
uncertainty as to the exact timing of up-regulation (between 8 and 11 days) and the
fact that half embryos appear to drop back in size after their catch-up may allow a
different explanation. That is, regulation may be some function of improved
nourishment via the visceral yolk sac, perhaps after the development of the yolk
sac's capillary network at around the 9th day (Snell & Stevens, 1966), and a
persistent inadequacy of the chorio-allantoic placenta may be responsible for the
subsequent decline in embryo growth when the true placenta becomes the primary
nutrient source.
I 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 their advice and encouragement.
This work was supported by a Medical Research Council Studentship.
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