J. Embryol. exp. Morph. Vol. 32, 2, pp. 495-503, 1974
Printed in Great Britain
495
Cell distribution in chimaeric mouse
embryos before implantation
By W E N D Y G A R N E R AND A N N E M c L A R E N 1
From the Department of Genetics, University of Edinburgh
SUMMARY
Eight-cell mouse embryos labelled by prior cultivation in the presence of tritiated thymidine
were aggregated with unlabelled 8-cell stages, and allowed to develop in vitro for two further
cell divisions. Autoradiographic analysis of the resulting embryos showed that little or no
mixing of the two cell populations took place during this period, either in the trophoblast or
in the inner cell mass.
INTRODUCTION
When two mouse embryos are aggregated at the 8-cell stage (Tarkowski, 1961)
the resulting adult chimaera (Mintz, 1965) shows evidence of extensive mixing
of the two cell populations. For example, the analysis of eye muscles each
derived from a single somite showed that, in chimaeric mice, all of 33 such
muscles contained cells from both components of the chimaera (Gearhart &
Mintz, 1972). How to interpret the distribution of the two cell populations in the
adult in terms of developmental processes in the embryo depends critically upon
when mixing of the two populations took place (McLaren, 1972). Little information on cell movement and cell mingling in the mammalian embryo is available
at present, even for the period of cleavage.
The scarcity of information reflects in part the difficulty of finding markers
suitable for following cell movements in early development. Mintz (1964«, b)
made aggregations between marked embryos, using either a difference in the
appearance of the cytoplasm or isotopic labelling of one embryo with tritiated
thymidine. She states that no specific pattern of cell distribution was evident in
the resulting blastocysts, but did not undertake any detailed analysis of the
material. We wished to get a picture of cell distribution after embryo aggregation, both in the outer trophoblast layer, and in the inner cell mass. For this
purpose the cytoplasmic-appearance criterion has limited application, as it
only applies to living material. We therefore labelled one component with
radioactive thymidine, allowed approximately two cell divisions to elapse after
aggregation, and examined the labelled population of nuclei on serial sections
by autoradiography.
1
Author's address: ARC Unit of Animal Genetics, Institute of Animal Genetics, West
Mains Rd., Edinburgh EH9 3JN, U.K.
32
E M B 32
496
W. GARNER AND A. MCLAREN
MATERIALS AND METHODS
Female mice of the randomly bred Q strain were killed on the afternoon of
the second day of pregnancy, and 2-cell embryos were flushed from the oviducts
with phosphate-buffered saline. The embryos were cultured for 24 h in drops of
modified Brinster's medium (Bowman & McLaren, 1970) under paraffin oil at
37 °C in a gas phase of 10 % C0 2 in air, with or without the addition to the
medium of [3H]thymidine (methyl-t-thymidine, specific activity 22-4 Ci/mmole,
Radiochemical Centre, Amersham). At the 8-cell stage the embryos were
treated with pronase to remove the zona pellucida, washed several times in
unlabelled medium, and cultured for a further 43 h in unlabelled medium either
as single embryos or as aggregated labelled-unlabelled pairs (Bowman &
McLaren, 1970). At the end of this period, the embryos were either air-dried
(Tarkowski, 1966), or transferred to mouse oviducts for ease of handling, fixed
in Carnoy's fluid, and wax-embedded. Serial 5 jum sections were cut, and autoradiographs prepared using AR-10 stripping film. Slides were developed after
14 days and stained with haematoxylin. To facilitate analysis of the embryos,
camera lucida drawings were made of every section, and labelled nuclei,
identified by the presence of silver grains (Fig. 1), were marked on the drawings.
RESULTS
Culture of mouse embryos in the presence of tritiated thymidine continuously
from the 2-cell to the blastocyst stage had been shown in this laboratory to give a
reduction in cell number at concentrations as low as 0-01 /tCi/ml (Snow, 1973).
Preliminary experiments were therefore carried out to determine a concentration
of [3H]thymidine which, if present from the 2-cell to the 8-cell stage, would give
adequate labelling without adversely affecting development. Culture from the
2-cell to the 8-cell stage in the presence of 0-1 /tCi/ml [3H]thymidine did not affect
the proportion of embryos subsequently forming blastocysts (91% of 45
embryos in medium containing [3H]thymidine, versus 87% of 31 embryos in
control medium), but significantly depressed cell number (Table 1). Lower
concentrations of [3H]thymidine did not affect cell number (Table 1). While
0-005 jLtd/ml gave an inconveniently low level of labelling, 0-01 /*Ci/ml
[3H]thymidine from the 2-cell to the 8-cell stage effectively labelled all the
embryonic cells up to the early blastocyst stage (32 cells). If the embryos were
allowed to develop to the late blastocyst stage, dilution of the label resulted in
low grain counts after autoradiography.
Embryos were therefore cultured in the presence of 001 /tCi/ml [3H]thymidine
for 24 h, from the 2-cell to the 8-cell stage, and then aggregated in pairs with
8-cell embryos which had been similarly cultured in unlabelled medium. The
aggregated pairs were cultured for a further 43 h in unlabelled medium before
histological processing and autoradiography. Table 2 gives cell counts carried
Cell distribution in chimaeric embryos
497
M
i.as^WÄ
/
y
/
f
B
.\r
j^W
7
Fig. 1. A-D are photographs of the first 4 serial sections of embryo 6. Nuclei 1-13
are numbered. All except 11 belong to trophoblast cells; 1, 6, 10, 11, 12 and 13
are labelled and 2, 3, 4, 5, 7, 8 and 9 are unlabelled.
out on sectioned material: the cell numbers of the single embryos, whether
labelled or unlabelled, are similar to those obtained from air-dried preparations
(Table 1), while the aggregated pairs contain approximately twice as many cells
as do single embryos.
The six labelled/unlabelled aggregants were analysed in detail, after serial
sectioning and autoradiography. Each cell was classified as labelled or unlabelled,
and as belonging to the trophoblast (cells contributing to the outer surface of
the embryo) or to the inner cell mass (wholly internal cells). As shown in Table 3,
the embryos gave consistent results : about one-third of all cells belonged to the
inner cell mass, and the proportion labelled was statistically homogeneous and
32-2
498
W. GARNER AND A. MCLAREN
Table 1. Cell counts of air-dried embryos grown from 2-cell to 8-cell in medium
containing various concentrations cf[3H]thymidine, and subsequently for 43 h in
unlabelled medium
Experiment
1
[3H]thymidine
concentration
(/tCi/ml)
0
0005
001
01
0
0005
001
No. of
embryos
Cell number
(mean ± standard error)
15
7
5
5
6
11
291 ±1-2
26-3 ±1-7
22-4±3-5
10-4 ±3-3
35-3 ±3-6
29-9 ±4-2
40-0 ±4-8
Table 2. Cell counts of sectioned embryos grown from 2-cell to 8-cell in the
presence of 0-01 /iCi/ml [3H]thymidine or in unlabelled medium, and subsequently
in unlabelled medium either singly or aggregated in pairs
2-cell to 8-cell
No of
embryos
Unlabelled
Unlabelled
Labelled
Labelled
5
6)
6/
11
Treatment
Cell number
(mean ± standard error)
Single
26-6 ± 2 0
Aggregated
64-7 ±3-7
Single
30-3 ±1-3
did not differ significantly from 50 % in either the inner cell mass or the trophoblast.
The distribution of labelled and unlabelled cells was examined separately for
the trophoblast, the trophoblast and inner cell mass interface, and the inner cell
mass.
The trophoblast on any section consists of a 1-cell-thick ring of cells. For a
ring of n cells, there are n adjacent pairs. To avoid the complications of considering the same cell twice, a set of disjoint sections was taken for each embryo,
in which no cell was represented more than once. Although not fully efficient,
this procedure is unbiased, and enabled 78 % of trophoblast cells to be included
in the analysis. For each section of the set, the observed numbers of like (both
unlabelled and both labelled) and unlike pairs were compared by x2 analysis
with those expected on a random-distribution hypothesis, using the proportions
of labelled and unlabelled cells in the set as a whole to calculate the expected
values. For a set of m sections, a total x2 tests deviations of any kind from the
null hypothesis (i.e. random distribution of labelled and unlabelled cells); the
overall X2D tests for an excess of like or unlike pairs in the embryo as a whole,
while the heterogeneity xfm-i) tests for differences between sections.
Cell distribution in chimaeric embryos
499
Table 3. Numbers of labelled and unlabelled cells in the trophoblast and
inner cell mass of six labelled/unlabelled aggregant embryos
Embryo
1
2
3
4
5
6
Total
°//o
Total no.
of cells
labelled
65
75
51
61
61
75
388
47-7
61 3
41-2
590
44-3
42-7
49-7
No. of
No. of cells iin
% of cells in
cells in
inner cell
inner cell
/o
/o
trophoblast labelled
mass
labelled
mass
47
50
35
38
39
46
255
44-7
660
45-7
39-5
53-8
391
48-6
18
25
16
20
21
29
129
55-6
520
31-3
56-5
27-3
48-3
53-5
27-7
33-3
31-4
32-8
34-4
38-7
33-2
Table 4. Analysis of the distribution of labelled and unlabelled cells in the
trophoblast of six labelled^ unlabelled aggregants
Embryo
No. of
disjoint
sections
Like
pairs/total
1
2
3
4
5
6
4
5
3
4
4
4
29/37
30/41
17/21
21/33
22/30
25/33
Xa)
Heterogeneity
(X2)
Mean no.
unlike pairs
per section (range)
12-6*
6-3*
80*
1-3
4-8*
80*
0-72
3-46
406
1300*
2-62
1-36
2 0 (2)
2-2 (0-4)
1-3(0-4)
3 0(0-8)
2 0 (0-4)
2-0 (2)
*P < 005.
If little mingling has occurred between the two aggregated embryos but the
interface is uneven, a significant heterogeneity x2 value will be expected if the
plane of sectioning of the embryo coincides approximately with the plane of
aggregation, and a significant overall x2 value if the plane of sectioning is at an
angle to the plane of aggregation. Table 4 shows that the null hypothesis is
contradicted for all six embryos ; embryo 4 appears to have been sectioned in the
plane of aggregation, and the others at an angle to it.
If no cell mingling has occurred, and either the interface between the aggregated embryos is even or the plane of sectioning is transverse to the plane of
aggregation, the expected number of unlike pairs per section will be 0 or 2
(Fig. 2 A). The last column of Table 4 shows that this expectation is fulfilled for
embryos 1 and 6. Embryos 2, 3 and 5 have end sections with cells of only one
type, and up to 4 unlike pairs in other sections. Inspection of the camera lucida
drawings suggests a plane of sectioning as in Fig. 2B. In embryo 4, one section
contains 8 unlike pairs out of 11, suggesting that the interface is very uneven and
the plane of sectioning is as in Fig. 2C.
To analyse the trophoblast-inner cell mass interface, each inner cell mass
500
W. GARNER AND A. MCLAREN
A
B
T
Fig. 2. Diagram of aggregated embryos with uneven interface but no cell mingling.
Lines represent histological sections. The expected number of 'transitions' in
each section will depend on the plane of sectioning.
Table 5. Analysis of the correspondence with respect to origin between inner cell
mass nuclei and adjacent trophoblast nuclei in six labelled <-> unlabelled aggregants
No. of
trophoblastinner cell
mass pairs
scored
Proportion
of
'like pairs'
Vd2 )
A
13
0-77
3-77
24
0-67
6-64*
14
20
21
25
071
0-70
076
072
2-18
3-20
6-53*
4-65*
* P < 005.
nucleus was paired with the trophoblast nucleus situated nearest to it on any
section on which it appeared. This procedure gives only an approximate estimate of the true spatial relations, especially for cells in the middle of the inner
cell mass or adjacent to the blastocoele. The pairs were again characterized as
'like' or 'unlike', and expected numbers of like and unlike pairs on a randomdistribution hypothesis were calculated from the observed numbers of labelled
and unlabelled trophoblast and inner cell mass cells in the embryo as a whole.
,\fi) tested departure from the random-distribution hypothesis for each embryo.
Table 5 shows that like pairs predominated in every embryo, with a highly
significant overall departure from random expectation.
Spatial relations in the inner cell mass proved still more difficult to evaluate
statistically, since the number of cells per section was small, each cell had
several neighbours, and cell boundaries could not be discerned with certainty.
A set of disjoint sections was again taken for each embryo, and the numbers of
labelled and unlabelled inner cell mass nuclei in each section were tabulated. If
the aggregated embryos remained largely discrete and the plane of sectioning
were more or less parallel to the plane of aggregation, there would be a tendency
for the ratio of labelled to unlabelled nuclei to change systematically from
Cell distribution in chimaeric embryos
Embryo 5
Embryo 6
Fig. 3. Diagrams of representative sections from embryos 1, 2, 5 and 6, in which
both labelled and unlabelled inner cell mass nuclei appeared on several sections.
The arrangement of labelled and unlabelled nuclei suggests that little cell mingling
has taken place. • , Unlabelled inner cell mass nuclei; 0> labelled inner cell mass
nuclei; • , unlabelled trophoblast nuclei; • labelled trophoblast nuclei.
501
502
W . G A R N E R A N D A. M C L A R E N
section to section. This was tested by a regression x2 analysis (Holt, 1948),
which proved statistically significant for embryos 3 (xfi) = 5-9) and 4 (xfi) = 7-6),
suggestive for embryos 1 (xfi) = 3-8) and 6 (xfi) = 3-1), and non-significant for
embryos 2 (x2i) = 0-1) and 5 (xfu = 00). In embryos 1, 2, 5 and 6 labelled and
unlabelled inner cell mass nuclei were both present on most of the sections, but
visual inspection suggested that little if any mingling had occurred. Fig. 3 shows
the arrangement of nuclei in representative sections from these four embryos.
DISCUSSION
The distribution of labelled and unlabelled cells in the aggregation chimaeras
described above does not support a hypothesis of random mixing between the
two cell populations. On the contrary, it seems that little if any mixing occurs in
either the trophoblast or the inner cell mass during the first two cell cycles after
aggregation, though some interdigitation of cells was seen at the aggregation
interface. These findings confirm the visual impression gained from time-lapse
cinematography of the development of chimaeric embryos up to the blastocyst
stage (Mulnard, 1971). They are also consistent with fig. 20 of Mintz (1964Û),
which shows a clump of four labelled nuclei in a blastocyst derived by aggregating six unlabelled 8-cell embryos with one labelled by prior incubation in 4 //Ci/
ml tritiated thymidine.
Hillman, Sherman & Graham (1972) have presented evidence to show that
individual cells of the mouse embryo are developmentally labile, at least up to
the 8-cell stage, and differentiate as trophoblast or inner cell mass in response to
a positional 'signal'. The results presented here support this view, since, if any
spatial pattern existed in the embryo at the 8-cell stage, one would expect to
see the pattern restored after aggregation by directed cell movement or reorganization between the two populations.
At some subsequent stage of embryonic development, extensive cell mingling
must occur, since in an adult chimaera most tissues contain representatives of
both constituent populations. The progeny of a single cell introduced at the
blastocyst stage may become widely distributed throughout the body (Gardner,
1971). For the inner cell mass, the mixing phase may begin during the downgrowth of the egg cylinder. Since trophoblast cells are already connected by
tight junctions at the early blastocyst stage, they are unlikely to shift their
relative positions significantly during the formation of primary trophoblast, but
mixing could well occur in the development of the ectoplacental cone.
We thank David Auerbach for his assistance during the early stages of this study, and the
Ford Foundation for financial support. W.G. was in receipt of an M.R.C. Studentship.
Cell distribution in chimaeric embryos
503
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{Received 14 February 1974)