/. Embryol. exp. Morph. Vol. 48, pp. 239-247, 1978
Printed in Great Britain © Company of Biologists Limited 1978
239
Investigation of the potency of cells
from the postimplantation mouse embryo by
blastocyst injection: a preliminary report
By J. ROSSANT, 1 R. L. GARDNER 2 AND H. L. ALEXANDRE 3
From the Department of Zoology, Oxford
SUMMARY
Chimaeric conceptuses have been produced by injection of 5£- and 6£-day extra-embryonic
ectoderm and 5£-day embryonic and extra-embryonic endoderm into 3|-day mouse blastocysts. Extra-embryonic ectoderm cells contributed only to the ectoplacental cone and/or
trophoblast giant cell fractions, reflecting the probable trophectoderm origin of these cells.
Proximal (visceral) endoderm cells overlying both the embryonic and extra-embryonic
ectoderm contributed cells only to the endoderm of the visceral yolk sac, indicating that the
definitive embryonic endoderm has not formed by 5£ days p.c.
INTRODUCTION
Recent manipulative studies have provided much information on cell fate and
potency in the preimplantation mouse embryo (reviewed Gardner & Papaioannou, 1975; Rossant & Papaioannou, 1977) and it is now desirable to extend
such studies into postimplantation development. However, the postimplantation
embryo is not readily amenable to manipulation since it is tightly embedded
within the uterus. Advances are being made in the culture of embryos over the
implantation period (e.g. Hsu, Baskar, Stevens & Rash, 1974; Wiley & Pedersen, 1977) but development is rarely normal and always limited. Until such
a culture system is entirely reproducible any manipulative experiments would
be difficult to interpret. Less direct approaches have so far been used to assess
postimplantation cell potency. In particular, studies have been carried out on
the growth of isolated tissues or tissue combinations in ectopic sites or in vitro
(reviewed Skreb, Svajger & Levak-Svajger, 1976; Rossant, 1977). However,
normal developmental potency may not be realized in these non-embryonic
environments (see Rossant, 1977). A further possible approach is to inject cells
1
Author's address: Department of Biological Sciences, Brock University, St Catharines,
Ontario, Canada, L2S 3A1.
2
Author's address (to whom all correspondence and reprint requests should be addressed):
Department of Zoology, South Parks Road, Oxford, 0X1 3PS, U.K.
3
Author's address: Department de Biologie Moleculaire, Universite Libre de Bruxelles,
67 Rue des Chevaux, 1640 Rhode-St-Genese, Belgium.
16-2
240
J. ROSSANT, R. L. GARDNER AND H. L. ALEXANDRE
Ectoplacental cone
Cut 2
ff
~Y\
Proximal (visceral)
.
. —.—;
{I
A
-4-4
Extra-embryonic endoderm
11
i\
\
^
\J
_|
Mural trophoblast giant cells +
Reichert's membrane +
parietal (distal) endoderm
Extra-embryonic ectoderm
Cut 1
Proximal (visceral)
Embryonic endoderm
Embryonic ectoderm
Fig. 1. Diagram of dissected 5^- or 6-day mouse egg cylinder to show position of cuts
and different tissues isolated.
from defined tissues of postimplantation embryos into blastocysts. Production
of chimaeras by this means could provide a powerful tool for analysing cell
commitment in this phase of development. Its chief advantage over the ectopic
graft or in vitro culture system is that the cells would be growing in an embryonic
environment so that normal cell-cell interactions could occur and the full range
of potential of a given cell should be expressed.
Injection of unspecified cells from 5£-, 8- and 12-day p.c. (post-coitum) mouse
conceptuses into blastocysts was undertaken earlier by Moustafa & Brinster
(1972). However, unequivocal chimaeras were only produced using blastocysts
recovered at 4 | days p.c. which were then cultured for a further 20-24 h before
being dissociated to provide cells for injection. The developmental status of
these embryos was not reported, but is unlikely to have been equivalent to that
of in vivo 5|-day p.c. embryos. The present study describes production of
chimaeric conceptuses after injection of two postimplantation tissues - extraembryonic ectoderm and proximal (visceral) endoderm - into 3-|-day blastocysts, and discusses the potential of this approach.
MATERIALS AND METHODS
Recovery of embryos
Host blastocysts were recovered on the 4th day after mating from mice of
a random-bred Swiss PO (Sir William Dunn School of Pathology, Oxford) or
CFLP stock (Anglia Laboratory Animals Ltd.), which were homozygous for
the a allele of glucose phosphate isomerase (Gpi-1&/Gpi-1&). Donor embryos,
which were Gpi-lh/Gpi-lh, were obtained from a variety of strains of mice. They
were dissected from the uterus on the 6th (5^-day embryos) and 7th (6|-day
embryos) day after mating. PBl + 10% foetal calf serum (Whittingham &
Wales, 1969) was used for all manipulations in this study.
Blastocyst injection of postimplantation cells
241
Isolation of postimplantation cells and injection into blastocysts
(a) Extra-embryonic ectoderm
Reichert's membrane with adhering trophoblast and parietal endoderm cells
was torn away from both 5|- and 6|-day embryos and the embryonic and
ectoplacental regions were cut away from the extra-embryonic regions using
glass microneedles (cuts 1 and 2, Fig. 1). Since no distinct division is discernible
between extra-embryonic ectoderm and ectoplacental cone an arbitrary cut 2
was made well below the insertion of Reichert's membrane (Fig. 1). The extraembryonic regions, consisting of extra-embryonic ectoderm plus overlying
endoderm, were then placed in 2-5 % pancreatin and 0-5 % trypsin in calcium,
magnesium-free Tyrode's saline (pH 7-6) at 4 °C for 10 min (Levak-Svajger,
Svajger & Skreb, 1969). Following this enzyme incubation the fragments were
returned to serum containing medium and the endoderm separated cleanly from
the extra-embryonic ectoderm by sucking the fragments up and down in
a flame-polished micropipette. The remaining lumps of extra-embryonic
ectoderm were then further divided by hand into small pieces of ca. 10-30 cells
with the aid of glass microneedles. These pieces were injected microsurgically
into the blastocoel of host blastocysts (Gardner, 1968, 1978). Blastocysts were
cultured for 1-2 h before transfer to the uteri of Gpi-1&/Gpi-1A recipients on the
3rd day of pseudopregnancy.
(b) Endoderm
Five and a half day embryos were dissected as above and the embryonic and
extra-embryonic regions were incubated separately for 10 min in a 0-25%
solution of trypsin in calcium, magnesium-free saline at 37 °C. Sucking up and
down in a micropipette produced a suspension of disaggregated cells from both
regions, in which the endoderm cells could be clearly recognized by their rough,
vesiculated appearance. Both embryonic and extra-embryonic ectoderm cells
have a smooth, rounded appearance in disaggregates (Gardner & Papaioannou,
1975). Between 6 and 10 endoderm cells isolated from either disaggregated
embryonic or extra-embryonic regions were injected into each of a series of host
blastocysts. Following a brief period in culture, the injected blastocysts were
transferred to Gpi-1&/Gpi-1& recipients on the 3rd day of pseudopregnancy.
Analysis of later development
(a) Extra-embryonic ectoderm
Recipients were killed at 9\ days of pregnancy and implants dissected into
four separate fractions: embryo ( + amnion), yolk sac, ectoplacental cone, and
trophoblast giant cells. The ectoplacental cone fraction consisted of overlying
giant cells, diploid trophoblast, chorionic and allantoic tissue. The giant cell
fraction consisted of the mural giant cells away from the ectoplacental cone
242
J. ROSSANT, R. L. GARDNER AND H. L. ALEXANDRE
from which the distal (parietal) endoderm layer and Reichert's membrane had
been cleared as much as possible. Separation of ectoplacental cone from mural
giant cells was not practicable in a few retarded embryos. The dissected samples
were treated and analysed electrophoretically for GPI by the method of
Chapman, Whitten & Ruddle (1971).
(b) Endoderm
Recipients were killed at 15| days of pregnancy and the conceptuses dissected
in phosphate-buffered saline into the following fractions; placenta, visceral
yolk sac, amnion and umbilical cord, liver, lungs, gut and the remainder of the
carcass. The yolk sac was further separated into its constituent endoderm and
mesoderm layers by prolonged cold pancreatin/trypsin incubation as described
elsewhere (Gardner & Rossant, in preparation). Any unseparated fragments of
the visceral yolk sac were retained as a separate fraction. The washed samples
were analysed for GPI as above.
RESULTS
Extra-embryonic ectoderm
Sixty-one conceptuses were obtained from blastocysts injected with pieces of
5-2-day extra-embryonic ectoderm tissue, 13 of which were found to be
chimaeric by GPI analysis. Nineteen chimaeras were found among 40 conceptuses developing from blastocysts injected with 6|-day extra-embryonic
ectoderm. The results of GPI analysis of these two sets of chimaeras are presented
in Tables 1 and 2. There was no case in which the injected extra-embryonic
ectoderm contributed to the embryo or to its yolk sac. Colonization was always
restricted to the ectoplacental cone and/or giant cell fraction. In the majority
of conceptuses, the injected cells contributed only to the giant cell fraction and
not detectably to the ectoplacental cone fraction. In some cases, e.g. C3, C5
( 5 | days, Table 1), the contribution of the injected cells to the giant cell fraction
was estimated to be as high as 50 %. Generally the level of colonization by the
injected cells was lower with 6^-day than 5|--day cells, presumably because of
the greater degree of temporal asynchrony involved. On the other hand, as
noted above, the actual frequency of chimaeras is greater for 6\- than 5-|-day
cells. This is most likely a technical artifact since it is much harder to isolate
the extra-embryonic ectoderm from 5\- than from 6|-day embryos.
Endoderm
The frequency of chimaeras was very low for 5|-day embryonic and extraembryonic endoderm. One hundred and three conceptuses were obtained after
injection of embryonic endoderm, of which only 12 were chimaeric. Four
chimaeras were found among 46 conceptuses developed from blastocysts
injected with extra-embryonic endoderm cells. In all 16 chimaeras the contribution of the injected cells was restricted to the endoderm of the yolk sac.
Blastocyst injection of postimplantation cells
243
Table 1. Distribution of GPI isozymes in chimaeric conceptuses derived from
blastocysts injected with 5\-day extra-embryonic ectoderm
Conceptus
code
no.
c2
c3
c4
Q
c.
c7
c8
c9
Cjo
Cu
C 12
cM
GPI analysis
Stage
Yolk Ectoplacental
Embryo sac
cone
Limb-bud
Limb-bud
Limb-bud
Limb-bud
Limb-bud
Limb-bud
Early limb-bud
Resorbing
Limb-bud
Limb-bud
Limb-bud
Resorbing
Limb-bud
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
Trophoblast
giant cells
A + B weak
A + B5:l
A
A
A
A + B weak
A + B weak
A + B 1:1
A + B weak
A+B 1:1
A + B 10:1
A + B weak
A
A
A
A + B2:
A
A + B5:
A
A + B5:
A
A+B 10:1
A + B 5:1
A + B weak
A + B weak
All conceptuses were analysed at 9£ days of pregnancy. The numbers next to the GPI
analysis for each fraction are visual estimates of the proportion of A to B isozyme.
Table 2. Distribution of GPI isozymes in chimaeric conceptuses derived from
blastocysts injected with 6\-day extra-embryonic ectoderm
Conceptus
code
GPI analysis
no.
Stage
C1
Limb-bud
Limb-bud
Resorption
Limb-bud
Limb-bud
Limb-bud
Limb-bud
Yolk sac only
Limb-bud
Limb-bud
Limb-bud
Limb-bud
Early limb-bud
Early limb-bud
Early limb-bud
Early limb-bud
Early limb-bud
Early limb-bud
Early limb-bud
c2
c3
c4
c5
c6
c7
c8
c9
c10
Cn
C12
c13
c14
c15
c16
c17
cC1918
A
t
Yolk Ectoplacental
cone
:Embryo sac
A
A
A
—
A
A
A
A
—
A
A
A
A
A
—
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
Trophoblast
giant cells
A + B 1:1
A + B weak
A
A + B2:l
A + B 10:1
A
A
A
A
A + B 10:1
A
A
A
A + B1:1
A+B 10:1
A + B 10:1
A + B2:l
A
A
A
A
A+B 20:1
A+B 20:1
A + B 15:1
A + B 15:1
A + B 10:1
A + B weak
A + B20:l
A + B weak
A + B 15:1
A + B weak
A+B 10:1
A + B 10:1
A + B 15:1
A + B 10:1
A + B weak
A + B5:l
All conceptuses were analysed at 9i days of pregnancy. The numbers next to the GPI
analysis i:or each fraction are visual estimates of the proportion of A to Bisozyme.
Yolk-sac
endoderm
A + B 10:1
A + B 10:1
A + B 10:1
A + B 2:1
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
Yolk-sac
mesoderm
A+B
A+B
A+B
A+ B
10:1
10:1
10:1
2:1
A + B 10:1
A
A + B 5:1
A + B5:l
A
A
A + B 15:1
A + B 10:1
A + B 10:1
A + B 15:1
A + B weak
A + B 20:1
Rest of yolk
sac
A
A
A
A
A + B 10:1
A
n.a.
A
A
A
A
A + B weak
A
A
A
A
Placenta
A
A
A
A
A
A
n.a.
A
A
A
A
A
A
A
A
A
Amnion +
umbilical
cord
A
A
A
A
A
A
n.a.
A
A
A
A
A
A
A
A
A
Liver
A
A
A
A
A
A
n.a.
A
A
A
A
A
A
A
A
A
Lungs
A
A
A
A
A
A
n.a.
A
A
A
A
A
A
A
A
A
Gut
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
Carcass
•
w
X
a
d
o
p
o
on
on
O
All conceptuses were analysed at 15+. days of pregnancy. The numbers next to the GPI analysis for each fraction are visual estimates of w
the proportion of A to B isozyme. n.a., not analysed.
c 15
C14
C-13
Extra-embryon ic
endoderm
Embryonic endoderm
A + B5:l
Q
A + B 10:1
C2
A + B 3:1
c3
Q
A + B5:l
Q
A + B2:l
Q
A + B 15:1
c7
A + B 10:1
c8
A + B2:l
Q
A + B 10:1
A + B2:l
A + B 20:1
Conceptus
code
number
GPI analysis
Table 3. Distribution of GPI isozymes in chimaeric conceptuses derived from blastocysts injected with 5\-day
embryonic and extra-embryonic endoderm
Blastocyst injection of postimplantation cells
245
The yolk-sac endoderm of C4 (Table 3) could not be analysed, but a chimaeric
contribution to the unseparated yolk sac and not to the separated mesoderm
indicates that the cells had colonized the endoderm. In no case was any contribution found in the endodermal organs of the foetus itself. Colonization of
the placenta was observed in two cases but this is not surprising since the yolksac endoderm is in close association with the placenta at the stage analysed.
DISCUSSION
Chimaeric conceptuses have been successfully produced by injecting 5\- and
6|-day extra-embryonic ectoderm and 5-|-day embryonic and extra-embryonic
endoderm cells into 3-|-day mouse blastocysts. These results attest once more
to the regulative capacities of the early mouse embryo since a considerable
degree of temporal asynchrony exists between donor and host cells. They also
establish that blastocyst injection is a feasible system with which to study the
potency of cells of the postimplantation embryo.
Some preliminary conclusions can be drawn about the potency of the particular cells injected in this study. The progeny of extra-embryonic ectoderm
cells contributed to the ectoplacental cone and/or the trophoblast giant cell
fractions but were never detected in the embryo or yolk-sac fractions. It is not
possible to identify the tissue or tissues being colonized in the ectoplacental cone
fraction, because the latter consists of both diploid and giant trophoblast,
chorion and allantois at 9-|- days p.c. However, nearly all chimaeras showed
a contribution, often large, by the injected extra-embryonic ectoderm cells to
the mural trophoblast giant cell fraction. It has been shown in blastocyst
'reconstitution' experiments that the trophoblast giant cell fraction derives
from the trophectoderm of the blastocyst and not the inner cell mass (Gardner,
Papaioannou & Barton, 1973). Thus, colonization of this fraction by injected
extra-embryonic ectoderm is consistent with the proposed trophectoderm
origin of the extra-embryonic ectoderm (Gardner & Papaioannou, 1975;
Gardner & Jonshon, 1975). It also confirms previous experiments which showed
that isolated extra-embryonic ectoderm retained the capacity to produce
trophoblast giant cells in ectopic grafts and in vitro (Rossant & Ofer, 1977).
The exact route by which extra-embryonic ectoderm colonizes the mural
trophoblast giant cell fraction after blastocyst injection is not clear. Nor is it
known (because of the composite nature of the ectoplacental cone fraction)
whether the injected cells ever actually contribute to the chorionic ectoderm,
which would be their normal fate in the intact embryo (Snell & Stevens, 1966).
It was hoped that both these questions might be answered by injecting rat
extra-embryonic ectoderm into mouse blastocysts and analysing the conceptuses
by immunofluorescence (Gardner & Johnson, 1973). However, no colonization
of mouse blastocysts by rat extra-embryonic ectoderm has been achieved
(Rossant & Johnson, unpublished). This failure is apparently not due to a funda-
246
J. ROSSANT, R. L. GARDNER AND H. L. ALEXANDRE
mental difference in the origin and properties of rat and mouse extra-embryonic
ectoderm as suggested by Mulnard (1974), since rat extra-embryonic ectoderm
isolated at 1\ and 8-| days of pregnancy also produces trophoblast giant cells in
ectopic sites (Rossant, 1977). Other approaches are now being used to provide
a more detailed analysis of chimaeras produced by injection of mouse
extra-embryonic ectoderm.
Both 5£-day embryonic and 5^-day extra-embryonic endoderm cells colonized
the visceral yolk-sac endoderm. Their progeny were never detected in any tissues
of the foetus itself, including its endodermally derived organs. An identical
result was found after injection of 4|-day primitive endoderm cells into blastocysts (Gardner & Papaioannou, 1975; Gardner & Rossant, in preparation),
from which it was concluded that the definitive endoderm of the foetus was
derived from the primitive ectoderm and not from the primitive endoderm. The
present result suggests that the definitive endoderm is still not represented in
the endoderm cell population by 5\ days. This is in agreement with ectopic
graft experiments in the rat which suggest that definitive endoderm does not
arise until the head-fold stage (Levak-Svajger & Svajger, 1974). The normal
fate of the endoderm in the embryonic region of the early egg cylinder is not
known (Rossant & Papaioannou, 1977). The present results demonstrate that it
has the potential to form visceral yolk-sac endoderm and may perhaps do so
normally.
The present experiments show that blastocyst injection can be useful in
studying postimplantation cell potency. However, this approach also has some
drawbacks. The frequency of chimaeras is low, particularly with endoderm
cells. In addition, the level of donor cell contribution to the resulting chimaeras
also tends to be rather low. In such cases, the full potential of the donor cells
might not be expressed in any one given chimaera. However, analysis of a number
of different chimaeras and selection of host and donor genotypes so as to maximize the donor contribution can overcome this problem. Finally, temporal
asynchrony between donor and host cells could also prove a problem with cells
from the later embryo. However, we feel that, given the current state of in vitro
experiments, the advantages of this method where cells are studied in an
embryonic environment outweigh its drawbacks.
We wish to thank Dr C. F. Graham and Mrs Joan Brown for help in preparing the
manuscript.
This work was supported by the Medical Research Council and the Royal Society. J.R.
was a Beit Memorial fellow. H.L. A. was supported by The Wiener-Anspach Foundation.
Blastocyst injection of postimplantation cells
247
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(Received 9 June 1978, revised 20 July 1978)