/ . Embryol. exp. Morph. Vol. 69, pp. 265-285, 1982
Printed in Great Britain © Company of Biologists Limited 1982
265
An autoradio graphic analysis
of tissue potency in different regions of the
embryonic ectoderm during gastrulation
in the mouse
By R. S. P. BEDDINGTON
From the Sir William Dunn School of Pathology
University of Oxford
SUMMARY
In vitro chimaeras have been produced by injecting [3H]thymidine-labelled 8th d;ay
embryonic ectoderm, derived from the anterior, distal or posterior regions of the egg cylinder,
into unlabelled synchronous embryos. Injected embryos were cultured for 36 h and the
distribution of donor cells was analysed autoradiographically. One series of orthotopic
injections was carried out and the results indicate that the developmental fate of embryonic
ectoderm in the posterior part of the embryo is to form mesoderm, both embryonic and
extraembryonic. Heterotopic injections of distal and posterior embryonic ectoderm demonstrate that these tissues readily conform to the colonisation patterns characteristic of their
new location. In contrast, anterior embryonic ectoderm showed some preference for definitive
ectoderm differentiation following heterotopic transplantation. However, there was no
evidence that the normal fate of tissue from the three regions studied could be explained by
pre-existing mosaicism in the embryonic ectoderm.
INTRODUCTION
There is now strong evidence in rodents that all the foetal primordia are
descended from a single population of cells, the embryonic ectoderm (LevakSvajger & Svajger, 1974; Gardner & Papioannou, 1975; Diwan & Stevens,
1976; Rossant, Gardner & Alexandre, 1978; Gardner & Rossant, 1979;
Beddington, 1981). During gastrulation the single epithelial sheet of embryonic
ectoderm is converted into a highly complicated form, made up of a variety
of tissue types and embodying the basic design of the foetus. This means that
the key to foetal organization must lie in the orderly allocation of tissue primordia within the embryonic ectoderm. The analysis of chimaeras produced
by orthotopic grafting of labelled embryonic ectoderm, at the late primitivestreak stage in the mouse, demonstrates that the allocation of tissues during
gastrulation follows a regular pattern (Beddington, 1981; see below), of, in
other words, that a fate map can be constructed. There are at least three possible
explanations for such a predictable topography of presumptive tissues:
266
R. S. P. BEDDINGTON
(i) Populations of embryonic ectoderm cells may have acquired differences in
their developmental potential such that their observed patterns of differentiation
in the embryo simply represent the fulfilment of their potential.
(ii) Embryonic ectoderm cells throughout the embryo may enjoy the same
potential but be subject to localized influences in the egg cylinder which effect
the selection of one developmental pathway, appropriate to a particular location,
at the expense of other possible ones.
(iii) Embryonic ectoderm cells may be heterogeneous in their developmental
potential, the regionalization of particular patterns of differentiation resulting
from the selection of cells with potential appropriate to that region.
In order to distinguish between these three possibilities it is necessary to
analyse the potential of embryonic ectoderm cells from different regions of the
egg cylinder and to compare this with their normal fate. The first two explanations can be tested at the tissue level whereas only a clonal analysis can discriminate between the second and third possibilities. This paper describes a
series of experiments designed to discriminate between the first two possibilities.
In vitro chimaeras have been used to analyse tissue fate and tissue potential
in the 8th day mouse egg cylinder. The normal fate of posterior embryonic
ectoderm was assessed following orthotopic injection. This extended previous
work on the developmental fate of anterior and distal embryonic ectoderm
(Beddington, 1981). In addition, the potency of anterior, distal and posterior
embryonic ectoderm was tested in heterotropic grafts. In these experiments it
was possible to establish whether the differentiation of donor tissue in a heterotopic site conforms to the colonization pattern characteristic of its new location
or whether it shows more identity with the normal fate of the donor cells.
MATERIALS AND METHODS
General strategy of the experiments
Tritiated thymidine-labelled embryonic ectoderm from three defined regions
of the 8th day embryonic egg cylinder was injected, either orthotopically or
heterotopically, into synchronous unlabelled host embryos. These injected
embryos were grown in culture for 36 h. Subsequently, the patterns of donor
cell colonisation were analysed autoradiographically. Embryos entirely labelled
with [3Hjthymidine, to the same extent as donor embryonic ectoderm tissue,
were also grown in culture for 36 h and processed for autoradiography (labelled
controls). These embryos provided a standard for the dilution of label in the
different foetal tissues over the culture period and also served as a control for
any inadvertant variation in the autoradiographic procedure.
Recovery and culture of embryos
All host and donor embryos were obtained from random-bred CFLP mice on
the morning of the 8th day of gestation. The embryos were dissected in PBI
38(100%.)
37(97-4%)
23(60-5%)
38(100%)
18(100%)
18(100%)
11(61-1%)
18(100%)
2(11-1%)
10-9 ±1-8
44(97-8%)
41 (911%)t
31(68-9%)
43 (95-6%)
6(13-3%)
9-9 ±2-7
4(10-5%)
10-4 ± 2 0
47
9(19-2%)
38(80-8%)
20
2(10%)
18(90%)
Distal
to
anterior
48
3(6-3%)*
45(93-7%)
Posterior
to
posterior
2(14-3%)
10-6 ±1-6
14(100%)
14(100%)
8(57-1%)
14(100%)
15
1(6-7%)
14(93-3%)
Posterior
to
anterior
1(5-6%)
10-1 ±1-5
18(100%)
17(94-4%)
10(55-6%)
18(100%)
20
2(10%)
18(90%)
Anterior
to
Distal
3(12%)
10-4 ±1-7
25(100%)
25(100%)
14(56%)
25(100%)
28
3(10-7%)
25(89-3%)
Posterior
to
Distal
3(10%)
100 ± 1 1
30(100%)
30(100%)
16(60%)
30(100%)
34
4(11-8%)
30(88-2%)
Anterior
to
Posterior
3(8-8%)
9-9 ±1-2
34(100%)
32(94-1%)
16(47-1%)
34(100%)
38
4(10-5%)
34(89-5%)
Distal
to
posterior
* Embryos retarded on the 8 th day of gestation were not selected for culture and, therefore, it is probably invalid to compare the incidence
of abnormal embryos recorded for in vivo controls with that for cultured embryos. It is likely that those embryos from in vivo which showed gross
abnormalities at the time of assessment may also have been retarded on the 8th day of gestation.
t The reduced number of in vivo controls showing a heartbeat and yolk-sac circulation is probably an artifact. During the time taken to dissect
a whole litter the heartbeat and/or yolk-sac circulation may cease.
% Somite numbers of the 7 classes of embryos receiving different injections do not differ significantly from one another, nor from those of in vivo
controls {t test; P > 005).
Total number
No. abnormal
No. normal
No. normal showing:
Heartbeat
Yolk sac
circulation
Turning
Trunk neural fold
fusion
Cranial neural fold
fusion
Somite number %
Nature of embryo
and injection
In vivo
control
(5 litters)
Table 1. A comparison of the development in culture of the seven classes of injected embryos and in vivo controls
Ov
to
1
I
Co
I
3
I
268
R. S. P. BEDDINGTON
medium (Whittingham & Wales, 1969) containing 10% (v/v) foetal calf serum
(FCS) instead of bovine serum albumin. Most of the parietal yolk sac, except
for the region adjoining the ectoplacental cone, was removed.
The roller system and the media used for culturing injected embryos and
labelled controls have been described in detail previously, and the efficiency of
the culture system together with the justification for using [3H]thymidine as a
single cell marker have also been discussed elsewhere (Beddington, 1981).
After removal from culture injected embryos and labelled controls were
compared with embryos of the same age which had been maintained in utero
until the end of the culture period (in vivo controls). This comparison included
both gross physiological and morphological characteristics (Table 1). Any
abnormal injected embryos were not included in the autoradiographic analysis of
chimaeras.
Labelling of donor embryos and labelled controls
After removal of Reichert's membrane, 8th day embryos were placed in
30 mm bacteriological dishes (Sterilin) containing a-medium (Flow Laboratories), to which had been added 10/*Ci/ml of [3H]thymidine (Radiochemicals,
Amersham) made up to a specific activity of 10-5 Ci/mM. The embryos were
cultured in this medium for 2 h at 37 °C in an atmosphere of 5 % CO2 in air.
During this period all the embryonic ectoderm nuclei become densely labelled
with [3H]thymidine (Beddington, 1981). After labelling the embryos were
washed for 15min in 3 changes of PBI plus 10% FCS supplemented with
10 fiu/ml of unlabelled thymidine.
Preparation and injection of labelled donor cells
One series of orthotopic injections was undertaken:
(1) (i) Injection of posterior embryonic ectoderm into the posterior region.
Six different heterotopic injections were carried out:
(2) (i) Heterotopic injections into the anterior region
(a) injection of distal embryonic ectoderm;
(b) injection of posterior embryonic ectoderm.
(ii) Heterotopic injections into the distal region;
(a) injection of anterior embryonic ectoderm;
(b) injection of posterior embryonic ectoderm.
(iii) Heterotopic injections into the posterior region:
(a) injection of anterior embryonic ectoderm;
(b) injection of distal embryonic ectoderm.
Figure 1 shows the location of the 3 regions used as a source of donor cells and
as the sites of injection.
After washing, labelled embryos were dissected with glass needles to isolate
Tissue potency in early postimplantation mouse embryos
=
269
Extraembryonic
ectoderm and
trophoblast
_ Visceral and
parietal endoderm
Embryonic and
= extraembryonic
mesoderm
Embryonic
ectoderm
Fig. 1. A drawing of a sagittal section through an 8th day embryo, as prepared for
culture, showing the three sites of injection and origin of donor tissue. A: Anterior;
D, distal; P, posterior.
the appropriate fragment of embryonic ectoderm. Anterior embryonic ectoderm was removed from beneath the foregut invagination and following removal
of excess lateral ectoderm consisted of a small rectangular piece of tissue
(approximately 70 x 50 /im). Distal embryonic ectoderm was removed from
the tip of the cylinder and again most of the lateral ectoderm was cut away
to leave a small square piece of tissue (approximately 70 x 70 /*m). Po$terior
embryonic ectoderm was dissected from the caudal end of the primitive Streak,
just beneath the origin of the afnnion, and trimmed to a size similar to that of
anterior embryonic ectoderm. Usually, contaminating endoderm and mesoderm
could be removed with glass needles but in some cases it was necessary to
subject the fragments to enzyme treatment, incubating them for.lOmin at
4 °C in a mixture of 0-5% trypsin and 2-5% pancreatin (Difco) in calciummagnesium-free Tyrode saline at pH 7-7 (Levak-Svajger, Svajger & Skreb,
1969). The ectoderm fragments were dissociated into small clumps of a size
suitable for injection using a very fine hand-drawn Pasteur pipette. These
clumps, consisting of about 20 cells (Beddington, 1981), were transferred to a
270
R. S. P. BEDDINGTON
micromanipulation chamber and injected into the selected region of a host
embryo using a micromanipulator assembly (Leitz) as described elsewhere
(Beddington, 1981).
Autoradiography
All injected embryos which were normal on recovery from culture, and at
least one labelled control in each experiment, were embedded in paraffin wax
(m.p. 56 °C) and serially sectioned at 5 fim. Wherever possible a labelled control
was included in the same wax block as injected embryos so that it would be
immediately obvious if the autoradiography processing was defective.
The wax sections were hydrated before being incubated in a 5 % solution of
trichloroacetic acid at 4 °C for 30 min. The slides were washed thoroughly in
running water and then placed in distilled water and transferred to a dark room.
Here they were covered with AR 10 fine-grain autoradiographic stripping film
(Kodak Ltd,) and left to expose at 4 °C for 3 weeks. They were developed using
D-19 developer (Kodak Ltd), and fixed in Kodafix solution (Kodak Ltd). Once
the slides were dry they were scanned in a light microscope to determine whether
or not the autoradiography was satisfactory. They were subsequently stained
with haemalum and eosin, or haemalum alone, mounted in DPX (BDH Chemicals Ltd.) and scanned carefully in a light microscope.
Injected embryos were only considered to be chimaeric if:
(1) They contained at least three labelled cells.
(2) Grains over a single nucleus were found in at least two consecutive sections.
(3) Labelled cells showed an equivalent grain density to that found over
the nuclei of the same tissue in labelled controls (cells were considered to be
dead if their nuclei were very densely labelled).
(4) Labelled cells were well integrated into embryonic tissues and were not
present as discrete unincorporated lumps.
The colonization patterns of donor tissue were recorded both in terms of the
particular tissues colonized and also according to the regional location of the
chimaerism. For this purpose the embryo was subdivided into three regions:
the head region (anterior to the first somite and including the heart; the trunk
region (that part which included all the somites); the caudal region (posterior
to the last somite). The number of colonizing cells in each chimaera was estimated by counting every labelled nucleus in alternate sections. Since the sections
were 5 ftm in width it was considered unlikely that a single nucleus would span
more than two sections. Indeed, the method of counting probably underestimates the actual number of colonising cells due to the low energy emission of
tritium particles and hence their short penetration distance (Rogers, 1973). A
labelled nucleus more than 2 ftm from the film may fail to cause silver grain
formation.
Tissue potency in early postimplantation
mouse embryos
271
Table 2. The rate of chimaerism following orthotopic and heterotopic injections
Site of
injection
Posterior
Anterior
Anterior
Distal
Distal
Posterior
Posterior
Nature of
donor cells
Posterior
Distal
Posterior
Anterior
Posterior
Anterior
Distal
No. of
embryos
screened by
No. with dead
autoradioNo. of chimaeras cells only
graphy
18 (0)*
25 (13)
14(0)
18(0)
23(2)
16 (14)
20 (14)
15(83-3%)
11 (44%)
9(64-3%)
12(66-7%)
14(60-9%)
8 (50%)
13(65%)
1(5-6%)
2(8%)
1(7-1%)
3 (16-7%)
2(8-7%)
6(37-5%)
2 (10%)
No. witty
unincor+
porated
lumps only
0
2(8%)
0
0
0
1(6-3%)
0
* The numbers in parentheses represent the number of normal embryos recovered from
culture which had to be excluded from the analysis due to defective autoradiographic processing.
RESULTS
Evaluation of injected embryos after culture
The developmental characteristics recorded for embryos from the seven
classes of injected embryos are shown in Table 1. These may be compared
with the same characteristics recorded for in vivo controls, which are also
represented in Table 1. Although there is some variation between the seven
classes of injected embryos in terms of the percentage of embryos in each class
which acquire certain characteristics, such as the fusion of cranial folds or
signs of axial rotation, there is no indication that any particular injection
consistently produces adverse effects on subsequent development. If the somite
numbers of each injected embryo class are compared separately with those
recorded for in vivo controls no significant differences are found (Mest; P ^
0-05). Similarly, the somite numbers of individual classes of injected embryos do
not differ significantly from one another (?-test; P ^ 0-05). The number of
normal injected embryos from each class which produced satisfactory autoradiographs is shown in Table 2. Exclusion of embryos from autoradiographic
screening was the result of whole batches of slides having to be discarded due
to defective processing. Table 2 also shows the number of chimaeras detected
in each class as well as the number of embryos containing only dead donor
cells or unincorporated lumps of donor tissue.
272
R. S. P. BEDDINGTON
Table 3. Distribution of colonizing cells following the injection of posterior
ectoderm into the posterior aspect of the primitive streak
Chimaera number
Dead cells
Unincorporated
lumps
No. incorporated
cells
Trunk surface
ectoderm
Trunk
neurectoderm
Trunk
loose mesoderm
Somite
Blood vessels
Midgut
Notochord
Caudal surface
ectoderm
Caudal
neurectoderm
Primitive streak
Caudal
loose mesoderm
Blood vessels
Hindgut
Notochord
1
2
3 4 5 6
7 8
9
10
ii
12
13
14
15
57
13
50
8
74
%
52 32 12 31 9 10 8 18 36 34
V
///,
YA
I 1i
if
ii i 1
i 1i I
Allantois
Blood cells
Amnion
Distribution of donor tissue in chimaeras
(1) Orthotopic injections
(i) Posterior embryonic ectoderm injected into the posterior region
All eighteen of the normal embryos injected with posterior ectoderm provided
satisfactory autoradiographs. The rate of chimaerism among these embryos is
high with fifteen embryos containing colonizing donor cells (83-3%). One
additional embryo (5-6 %) contained only dead cells of donor origin (Table 2).
The pattern of colonization was very consistent among these embryos with the
distribution of labelled cells being restricted to embryonic and extraembryonic
mesodermal tissues (Table 3). Most of the chimaeras contained some dead cells
of donor origin although no unincorporated lumps were detected. All of the
chimaeras except one contained labelled cells in the loose mesoderm of the
caudal region and in nine of these embryos (nos. 2, 4-9 and 13) the endothelial
lining of the blood vessels, either arteries or veins, had been colonized in this
region (Table 3). Three embryos were chimaeric in somite mesoderm, but only
the most recently formed or penultimate somite had been colonized. The
Tissue potency in early postimplantation mouse embryos
273
Table 4. The distribution of colonizing cells in chimaeras injected in the anterior
region with distal ectoderm
Chimaera number
Dead cells
Unincorporated lumps
No. colonizing cells
156
93
117
39
187
163
100
57
20
Head surface ectoderm
Head neurectoderm
Head loose mesoderm
Heart
Blood vessels
Foregut
allantois was chimaeric in seven embryos (nos. 8-13 and 15) and in all but one of
these chimaeras (no. 15) labelled cells in the allantois were contiguous with those
in the posterior embryonic mesoderm. The distribution of donor cells was, in
general, quite diffuse, labelled cells bejng interspersed among host cells with
seldom more than three donor cells lying directly adjacent to one another.
However, in blood vessels donor cells showed greater coherence in their
distribution.
(2) Heterotopic injections
(i) The anterior region
{a) Injection of distal embryonic ectoderm. Thirty-eight normal injected
embryos were recovered from culture following the injection of distal ectoderm
into the anterior region. Satisfactory autoradiographs were obtained for 25 of
these embryos. Two embryos contained only heavily labelled dead cells and a
further two embryos had unincorporated lumps of labelled cells in the amniotic
cavity (Table 2). Eleven (44 %) of the remaining embryos were classified as
chimaeras (Table 2). The distribution of colonizing donor cells in the chimaeras
is shown in Table 4. Only three tissues had been colonized: head surface
ectoderm, head neurectoderm and heart mesoderm. Most of the embryos
exhibited surface ectoderm chimaerism (chimaeras nos. 1-7). Four chimaeras
(nos. 6-9) had been colonized in the neurectoderm and of these two contained
labelled cells in the adjacent surface ectoderm (nos. 6 and 7). Three embryos
were chimaeric in the epimyocardial component of the heart (nos. 5, 10 and 11)
and one of these (no. 5) had a very small separate patch of colonizing cells
(7 cells) in the surface ectoderm. In all cases the colonizing cells were foun^ in
coherent patches suggesting that the cells remained together after grafting &nd
that there was little cell mixing with host tissue. Six of the chimaeras contained
additional dead donor cells either adhering to the amnion or to the surface of
the embryo.
274
R. S. P. BEDDINGTON
Tissue potency in early postimplantation mouse embryos
Table 5. The distribution of colonizing cells in chimaeras
injected in the anterior region with posterior ectoderm
1
Chimaera number
2
3
4
5
n
Dead cells
Unincorporated lumps
No. colonizing cells
Head surface ectoderm
Head neurectoderm
68
45
55
6
7
nWW m
10
H9,w,
12 43
43 69 23
66
9
8
ii
^§
41
50
mW.
w,
WA
Head loose mesoderm
b
Heart
Blood vessels
Foregut
Table 6. 77ie distribution of colonizing cells in chimaeras injected
in the distal region with anterior ectoderm
Chimaera number
Dead cells
Unincorporated lumps
No. colonizing cells
Trunk surface ectoderm
Trunk neurectoderm
Trunk loose mesoderm
Somite
Blood vessels
Midgut
Notochord
1
2
3
4
5
6
1
8
9
10
H
7 23 23
11 12
WA
8
7
20 42
W/<
37
11
33
18
6
WA
Posterior surface
ectoderm
Posterior neurectoderm
Primitive streak
11H
Posterior loose mesoderm
Blood vessels
Hindgut
Notochord
Fig. 2. Transverse sections through chimaeras. (A) Chimaera no. 1. (anterior embryonic ectoderm injected into the posterior region). (B) Chimaera no. 7. (posterior
embryonic ectoderm injected into the anterior region). (C) Chimaera no. 2 (posterior
embryonic ectoderm injected into the anterior region). (D) Chimaera no. 3. (posterior embryonic ectoderm injected into the anterior region). NE, neurectoderm;
SE: surface ectoderm; M: mesoderm; FG, foregut; AM: amnion. Donor cells are
marked by arrows. Bar = 40 /tm.
275
276
R. S. P. BEDDINGTON
Table 7. The distribution of colonizing cells in chimaeras injected
in the distal region with posterior ectoderm
Chimaera number
1
Dead cells
Unincorporated
lumps
No. incorporated
cells
i
Trunk surface
ectoderm
Trunk
neurectoderm
Trunk loose
mesoderm
Somite
Blood vessels
Midgut
Notochord
Table 8.
3 4
%
5
6 7
8
9 10
i
%,
12
13
14
23
12
13
^ ^
10 74 32 25 18 19 51 14 11 35
F
ii
16
i i i i i 1I i
# ^
distribution of colonizing donor cells in chimaeras injected in the
posterior region with anterior ectoderm
Chimaera number
Dead cells
Unincorporated lumps
No. colonizing cells
52
56
32
34
18
54
8
23
Posterior surface ectoderm
Posterior neurectoderm
Primitive streak
Posterior loose mesoderm
Blood vessels
Hindgut
Notochord
Allantois
Blood cells
Amnion
(b) Injection of posterior embryonic ectoderm. The injection of posterior
ectoderm into the anterior region generated fourteen normal embryos all of
which provided satisfactory autoradiographs. Nine embryos qualified as
chimaeras (64-3 %) and one other embryo showed the presence of dead cells in
the amniotic cavity (Table 2). From the distribution of donor cells in the chimaeras (Table 5; Fig. 2B-D) it is clear that chimaerism predominates in the
Tissue potency in early postimplantation
mouse embryos
277
Table 9. The distribution of colonizing donor cells in chimaeras injected in the
posterior region with distal ectoderm
surface ectoderm (nos. 1-8). Four of these embryos also showed colonisation
of the anterior extreme of the foregut (nos. 1-4) and in all four cases this labelled
region was continuous with the labelled surface ectoderm and, therefore, was
considered to be in the ectodermal component of the foregut, the stomodeiim
(Fig. 2D). One embryo (no. 11) contained donor tissue in the epimyocardium
of the heart. Once again labelled colonizing cells occurred only in discrete
coherent patches and there was little indication of mixing with host cells. Five
of the chimaeras contained dead donor cells in addition to colonizing cells.
278
R. S. P. BEDDINGTON
Table 10. A summary of the tissue distribution of colonizing cells in each series
of orthotopic and heterotopic injections
t A: anterior region; D: distal region; P: posterior region
* Results taken from Beddington (1981).
% Chimaerism at the anterior extreme of the foregut has been classified as surface ectoderm
colonization (see p. 277).
• 50-100% chimaeras colonized in that tissue.
B3 20-49 % chimaeras colonized in that tissue.
[H Less that 20 % chimaeras colonized in that tissue.
(ii) The distal region
(a) Injection of anterior embryonic ectoderm. Eighteen normal embryos, which
had received injections of anterior ectoderm into the distal region, were processed
satisfactorily for autoradiography. Twelve of these embryos proved to be
chimaeric (66-7 %) and a further three embryos showed evidence of dead donor
cells in the amniotic cavity (Table 2). The distribution of colonizing cells is
shown in Table 6. In all the chimaeras it was only neurectoderm tissue which
had been colonized. In nine of the chimaeras colonization was restricted to the
trunk region (nos. 1-9) but in one case (no. 10) labelled cells were found extending from the trunk region into the posterior part of the embryo. In the two
other chimaeras (no. 11 and 12) only caudal neurectoderm had been colonized.
Labelled cells were not scattered throughout the neural tube or neural plate
but were present as distinct patches within the neurectoderm. Dead donor cells
were present in six of the chimaeras.
(b) Injection of posterior embryonic ectoderm. Twenty-five normal embryos
were obtained from injections of posterior ectoderm into the distal tip of the
egg cylinder. Two of these were lost during embedding but the remaining
twenty-three provided satisfactory autoradiographs. Fourteen of these embryos
(60-9%) were chimaeric and a further two embryos contained dead cells in
the amniotic cavity (Table 2). Colonizing donor cells were present only in
mesodermal tissues (Table 7). The majority of chimaeras had been colonized
Tissue potency in early postimplantation mouse embryos
279
in the trunk loose mesoderm (nos. 1-10) and seven of these showed donor cells
lining the caudal artery (nos. 1-7) and one (no. 10) was also chimaeric in
somites on one side of the neural axis. Four other embryos were colonized
exclusively in somites, two showing bilateral colonization (nos. 12 and 13) and
two only unilateral colonization (nos. 11 and 14). Chimaerism was restricted tQ
the trunk region. In blood vessels the donor cells tended to occur as coherent
patches whereas in the loose mesoderm and somites, although patches were
apparent, the donor cells were intermingled with host cells. Nine chimaeras
contained an additional population of densely labelled dead cells.
(iii) The posterior region
(a) Injection of anterior embryonic ectoderm. Thirty normal embryos, which
had received injections of anterior ectoderm into the posterior aspect of the
primitive streak, were processed for autoradiography. Due to slipping of the
autoradiographic film only sixteen of these could be screened for chimaerism.
Eight embryos were classified as chimaeras (50%). A further six embryos
contained only dead labelled cells and one additional embryo had a lump of
unincorporated donor tissue attached to the caudal vein (Table 2). Two of the
chimaeras (nos. 1 and 2) were colonized only in the surface ectoderm of the
caudal region (Table 8; Fig. 2A). One chimaera (no. 3) contained donor celis
in both the caudal surface ectoderm and adjacent mesoderm. Four embryos
(nos. 4-7) were colonized only in the caudal loose mesoderm and the last
chimaera (no. 8) was colonized only in the allantois. Donor cells in the surface
ectoderm occurred as coherent patches whereas in the mesoderm and the allafitois they had a more scattered distribution. Chimaerism was restricted to the
posterior region of the embryo except in one case where extraembryonjc
mesoderm had been colonized. Densely labelled dead cells were evident in the
majority of chimaeras, both within the embryonic tissues and in the amniotic
cavity.
i
(b) Injection of distal embryonic ectoderm. Twenty normal embryos, injected
with distal ectoderm, provided satisfactory autoradiographs. Thirteen of these
embryos were chimaeric (65 %) and a further two embryos contained only de^d
cells (Table 2). All except three chimaeras (nos. 11, 12 and 13) contained donor
cells in the caudal loose mesoderm (Table 9). The allantois was colonized in all
but two of the chimaeras (nos. 8 and 13). Three embryos had been colonized
in the caudal vein (nos. 1-3) and one of these also had donor cells in the endothelial lining of the caudal artery (no. 1). Chimaera no. 4 had labelled cells
lining the caudal artery in the trunk region. One embryo (no. 13) had been
colonized in the mesodermal component of the amnion adjacent to the lateral
limiting sulcus. Labelled cells showed a scattered distribution in the allantois and
loose mesenchyme indicating that some mixing with host cells had occurred.
The colonization of blood vessels tended to be rather sparse but in one chimaera
(no. 4) nineteen cells were found lining the caudal artery in the trunk region and
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R. S. P. BEDDINGTON
they occurred as a coherent patch. Nine of the embryos contained additional
dead donor cells and one embryo (no. 6) had a lump of unincorporated donor
tissue stuck to the mesoderm layer of the yolk sac.
DISCUSSION
Tissue fate
In a previous study (Beddington, 1981) the developmental fate of anterior
and distal embryonic ectoderm was analysed using an orthotopic grafting
technique. An identical procedure has been used here to investigate the fate of
posterior embryonic ectoderm. The overall pattern of tissue fate which emerges
is as follows (Table 10):
(i) anterior embryonic ectoderm gives rise to predominantly definitive ectoderm
derivatives: surface ectoderm and neurectoderm;
(ii) distal embryonic ectoderm generates definitive gut endoderm, notochord
and embryonic mesodermal derivatives, including somites;
(iii) posterior embryonic ectoderm contributes exclusively to mesodermal
tissues, both embryonic and extraembryonic.
It is clear, therefore, that different regions of the embryonic ectoderm give
rise to different tissues. Such regularity in tissue allocation is not unexpected
considering the well-documented fate maps, constructed during gastrulation,
for embryos from other animal classes. What is, perhaps, significant is that
these rudiments of a fate map for the mouse primitive-streak-stage embryo fit
the general scheme of presumptive tissue topography found throughout the
chordate phylum at an equivalent developmental stage (Pasteels, 1937; Nicolet,
1971). It remains to be seen whether such a consistent pattern of tissue fate
reflects a common method of tissue allocation, or instead, is simply a 'preview'
of similarities in subsequent morphogenetic mechanisms and tissue interactions
essential to the initial development of the organ rudiments.
Tissue potential
The heterotopic series of injections provides no evidence for rigid mosaicism
in the embryonic ectoderm tissue during the later stages of gastrulation. Both
distal and posterior embryonic ectoderm readily colonize surface ectoderm
when placed in an anterior site (Table 10) despite the fact that this tissue is
never colonized following their orthotopic injection (Table 10; Beddington,
1981). Similarly, embryonic ectoderm from all three regions shows the capacity
to form a variety of mesodermal derivatives in heterotopic sites. However,
although anterior embryonic ectoderm will colonise both embryonic and
extraembryonic mesoderm when relocated in the posterior region, it does show
a preference for definitive ectoderm differentiation wherever it is placed in the
embryo (Table 10). Furthermore, neither anterior nor posterior embryonic
ectoderm gave rise to gut endoderm or notochord in any experiment. Endoderm
Tissue potency in early postimplantation mouse embryos
281
and notochord colonization was obtained only after orthotopic distal grafts
(Beddington, 1981).
The ability of embryonic ectoderm outside the presumptive definitive ectoderm area (the anterior region) to form definitive ectoderm derivatives is
consistent with studies on other vertebrate embryos. For instance, if the
blastopore lip from an amphibian gastrula is transplanted to the presumptive
ectoderm area it will form epidermal and neural structures (see Holtfreter &
Hamburger, 1955). Chorioallantoic grafts of different regions of the chick
blastoderm, at the head process stage, demonstrate that almost the entire
blastoderm, and almost certainly only the epiblast component of it, retains
the ability to form epidermis (see Waddington, 1952). In addition, experiments
on primary induction in both amphibian (Spemann, 1938) and avian (Waddington, 1952; Gallera, 1971) embryos demonstrate that regions of the embryo
normally destined to form other tissues are competent to produce neural
structures. A comparable pluripotency is found during gastrulation with respect
to mesodermal differentiation. Both amphibian (Holtfreter & Hamburger,
1955) and avian (Waddington, 1952) prospective definitive ectoderm can form
any sort of mesodermal tissue. Therefore, with regard to epidermal, neural arid
mesodermal differentiation the fate maps in amphibian and avian grastrulae
cannot be explained by any pre-existing mosaicism of developmental potential
in different prospective tissues. The same would seem to be true of the primitivestreak-stage mouse embryo.
However, one cannot ignore the tendency of anterior embryonic ectoderm to
form definitive ectoderm derivatives in heterotopic sites. There is no ready
explanation for this behaviour, although it has been found in chick embryos, at
a similar stage, that grafts of prospective definitive ectoderm show a certain
reluctance to invaginate and that, in the absence of invagination, only neural
structures are formed (Waddington & Taylor, 1937; Abercrombie, 1937). A
similar failure to invaginate may account for the neural differentiation of
anterior embryonic ectoderm in the distal region and its colonization of surface
ectoderm in the posterior region. One might speculate that tissue fate becomes
stabilized first in the anterior region and that this stabilization is associated
with a resistance to any breakdown in epithelial organization. Some support
for this idea comes from recent work on experimental teratomas. Pre-primitivestreak rat embryonic ectoderm rapidly loses its epithelial organization when
grafted beneath the kidney capsule, whereas headfold-stage ectoderm, while
producing some mesoderm, shows a greater tendency to maintain its epithelial
structure (Svajger, Levak-Svajger, Kostovic-Knezevic & Bradamante, 1981),
Chimaerism in the gut and notochord occurs only following distal orthotopic
grafts. This might suggest that the potential to generate gut and notochord is
restricted to distal embryonic ectoderm. Such an interpretation would be
consistent with experiments in birds. In chorioallantoic grafts of the chick
embryo at the head-process stage only the anterior part of the primitive streak
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R. S. P. BEDDINGTON
will give rise to notochord, although the ability to form gut is more widespread
(Waddington, 1952). Furthermore, studies on primary induction, using either
[3H]thymidine or the quail nucleolar marker to distinguish the grafted Hensen's
node, demonstrate that the gut and notochord in induced secondary axes are
invariably of graft origin (Gallera & Nicolet, 1969; Hornbruch, Summerbell &
Wolpert, 1979). This indicates that in avian embryos the differentiation of
notochord, and to a lesser extent gut, is associated specifically with the anterior
part of the primitive streak, or Hensen's node. However, grafts of mouse distal
embryonic ectoderm, presumed to be equivalent to Hensen's node, show no
inclination for autonomous differentiation, as judged by their failure to produce
gut and notochord in heterotopic sites. This could be explained by the small
size of the grafts (~ 20 cells) compared with those used in chick experiment. A
more important discrepancy is that anterior embryonic ectoderm readily
produced definitive endoderm derivatives in experimental teratomas (R. S. P.
Beddington, in preparation). Therefore the formation of gut and notochord in
the mouse embryo cannot simply be attributed to a strict mosaicism in developmental potential. Certain influences or morphogenetic requirements peculiar
to the distal region must also be important. However, while it is clear that distal
embryonic ectoderm can respond to these cues, anterior and posterior embryonic ectoderm apparently cannot. One must conclude that the formation of
gut and notochord in the embryo involves a rather precise interplay between
the competence to form these two tissues and the ability to respond to certain
cues in the distal region.
The results demonstrate that 8th day embryonic ectoderm does not behave
as a mosaic tissue consisting of strictly demarcated areas, each destined to form
a particular tissue by virtue of its appropriate restriction in developmental
potential. This is contrary to the conclusions drawn by Snow (1981) based on
experiments in which particular fractions of the embryonic egg cylinder, usually
consisting of all three germ layers, differentiated in an apparently autonomous
fashion when isolated in vitro. This was considered to be indicative of mosaic
development. However, the autonomous differentiation of the fractions cannot
be interpreted as a reflection of mosaicism in the embryonic ectoderm since
most isolates also contained mesoderm and all of them had an endodermal
component. The presence of other tissues is very likely to affect the development
of embryonic ectoderm. For example, it is well known that the type of neural
structure formed by amphibian ectoderm is dependent on the inducer and not
an inherent property of the ectoderm (see Holtfreter & Hamburger, 1971;
Toivonen & Saxen, 1968). Therefore, although regionalization may be apparent
in explants where the germ layer relationships are not disturbed this does not
mean that there is necessarily any regionalization in developmental potential
within the embryonic ectoderm.
Tissue potency in early postimplantation mouse embryos
283
Patterns of growth during development
The distribution of labelled cells in the various tissues of the chimaeras, froiti
both orthotopic and heterotopic injections, provides some indication of the
patterns of growth within those tissues at the onset of organogenesis. For
example, labelled cells were always found in well defined patches in the surface
ectoderm, neurectoderm, notochord, gut and endothelial lining of blood
vessels. In contrast, the distribution of labelled cells in certain mesodermftl
derivatives, such as lateral plate mesenchyme and the allantois, was mofe
diffuse.
Patterns of growth and the degree of cell mixing during development have
been studied extensively in mouse chimaeras and mosaics (West, 1978). Autoradiographic analysis of aggregation chimaeras at the blastocyst stage has shown
that there is little or no cell mixing during cleavage and blastocyst formation
(Garner & McLaren, 1974). However, if only a few cells (2-3 cells) from a 4th
day embryo are injected into the blastocyst their progeny can be detected in
very small samples of every adult tissue examined (Ford, Evans & Gardner,
1975). In addition, the injection of single 5th day embryonic ectoderm cells
generally results in chimaerism throughout the foetus and also in the extfaembryonic mesoderm (Gardner & Rossant, 1979). As prospective foetal tissues
appear to have a definite topographical arrangement at the late primitive-streak
stage this must mean either that there is considerable cell mixing in the embryonic ectoderm prior to gastrulation or that growth in the egg cylinder occurs
such that descendant clones tend to be aligned with, and extend along, the
length of the embryonic axis. If this were not the case, one would expect the
clonal descendants of a primitive ectoderm cell injected at the blastocyst st&ge
to be contained within just one or two prospective areas, and, therefore, to give
rise only to a limited number of foetal or adult tissues. Instead it seems that
progeny of a single primitive ectoderm cell must be distributed so that they are
present in all prospective areas, ranging from the anterior region of the egg
cylinder (prospective surface ectoderm and neurectoderm) to the posterior end
of the primitive streak (prospective embryonic and extraembryonic mesodefm).
It is interesting that growth in the surface ectoderm and neurectoderm appears
to be coherent at the onset of organogenesis (see above). The analysis of p&tch
size in the epidermis of adult mouse chimaeras, using electrophoretic allozyme
variants, reveals that fragments of skin less than 1 mm2 in area nearly always
contain cells of both host and donor origin (Iannacone, Gardner & Harris, 1978).
This indicates that the final patch size in the epidermis is very small, and therefore, extensive cell mingling must occur after the initial delamination of the
surface ectoderm. Similarly, the estimated coherent clone size in the retinal
pigment epithelia, a neurectoderm derivative, in both mouse chimaeras and
mouse X-inactivation mosaics, on the 13th day of gestation is extremely sfliall,
constituting no more than one .or two cells (West, 1978). Although there is
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R. S. P. BEDDINGTON
evidence that the distribution of cells is not entirely random at this stage,
since descendant clones of one phenotype tend to be clustered into sectors
(Sanyal & Zeilmaker, 1977), the distribution once again points to considerable
cell mixing among neurectoderm cells prior to the foundation of the retina
pigment epithelia.
In conclusion, it appears that clonal growth during mouse development
must alternate between being coherent and involving extensive cell mingling.
During cleavage and blastocyst formation growth is coherent but after implantation there must be cell mixing, at least in the embryonic ectoderm. At the onset
of organogenesis much of the earliest differentiation and morphogenesis of the
germ layers appears to be associated with coherent growth but, once again, this
seems to be followed by a phase of cell mixing before the final allocation of the
different organ primordia.
I would like to thank Professor R. L. Gardner, Dr V. E. Papaioannou and Miss G. Porter Goff for invaluable discussion. This work was supported by a Medical Research Council
Studentship.
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(Received 14 December 1981, revised 18 January 1982)
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