/ . Embryol. exp. Morph. Vol. 68, pp. 175-198, 1982
Printed in Great Britain © Company of Biologists Limited 1982
Investigation of cell lineage and differentiation
in the extraembryonic endoderm of the
mouse embryo
ByR. L. GARDNER1
From the Sir William Dunn School of Pathology, University of Oxford
SUMMARY
The technique of injecting genetically labelled cells into blastocysts was used in an attempt
to determine whether the parietal and visceral endoderm originate from the same or different
cell populations in the early embryo. When the developmental potential of 5th day primitive
ectoderm and primitive endoderm cells was compared thus, only the latter were found to
colonize the extraembryonic endoderm. Furthermore, single primitive endoderm cells yielded
unequivocal colonization of both the parietal and the visceral endoderm in a proportion of
chimaeras. However, in the majority of primitive endodermal chimaeras, donor cells were
detected in the parietal endoderm only, cases of exclusively visceral colonization being rare.
Visceral endoderm cells from 6th and 7th day post-implantation embryos also exhibited a
striking tendency to contribute exclusively to the parietal endoderm following blastocyst
injection.
The above findings lend no support to a recent proposal that parietal and visceral endoderm are derived from different populations of inner cell mass cells. Rather, they suggest that
the two extraembryonic endoderm layers originate from a common pool of primitive endoderm cells whose direction of differentiation depends on their interactions with non-$ndodermal cells.
INTRODUCTION
Primitive endoderm cells appear to differentiate on the blastocoelic surface of
the inner cell mass (ICM) and, according to Nadijcka & Hillman (1974), can
usually first be discerned by electron microscopy before the zona pellucida is
lost, late on the fourth day of development. By the middle of the fifth day they
can be distinguished from the remaining primitive ectoderm cells in dissociated
ICMs, thereby enabling one or more cells of either type to be injected into
genetically dissimilar host blastocysts (Gardner & Rossant, 1979). Following
transplantation, primitive endoderm cells were found to colonize only one of the
ICM derivatives into which host conceptuses were dissected, namely the extraembryonic endoderm of the visceral yolk sac. Primitive ectoderm cells, in
contrast, contributed to all foetal and extraembryonic fractions of ICM origin
that were analysed, except the visceral yolk-sac endoderm. Indeed, the precision
1
Author's address: Sir William Dunn School of Pathology, University of Oxford, South
Parks Road, Oxford 0X1 3RE U.K.
176
R.L.GARDNER
* Parietal endoderm
Primitive endoderm
Visceral endoderm
Early ICM
\
Extraembryonic mesoderm
Primitive ectoderm ^ — • Amniotic ectoderm
Foetus
Parietal endoderm
^
/
Primitive ectoderm
-£-
Visceral endoderm
Extraembryonic endoderm
Z — Amniotic ectoderm
Foetus
Fig. 1. Alternative cell lineages for the origin of the parietal and visceral endoderm.
(A) represents the scheme proposed by Gardner & Papaioannou (1975) and (B) the
scheme proposed by Dziadek (1979).
with which the two classes of cells partitioned in host embryos suggested that
both had acquired a relatively stable state of differentiation by the late blastocyst stage (Gardner & Rossant, 1979).
However, several studies on isolated lCMs grown in vitro have led to the
conclusion that the primitive ectoderm retains the option of forming extraembryonic endodermal cells after endodermal differentiation has taken place
(Pedersen, Spindle & Wiley, 1977; Atienza-Samols & Sherman, 1979; Dziadek,
1979). This is based on the finding that if the layer of endoderm which differentiates initially on the surface of such ICMs is destroyed by immunosurgery,
the purportedly endoderm-free cores of primitive ectoderm cells left behind can
generate a new one. Dziadek (1979) found, in addition, that the regenerated
endoderm differed from the original layer in displaying a much higher proportion
of cells that reacted with an antiserum directed against alpha-foetoprotein,
a visceral endoderm cell marker (Dziadek & Adamson, 1978). This led her to
propose that two distinct phases of extraembryonic endoderm differentiation
take place normally during development; formation of an initial parietal cell
layer being succeeded by production of a second visceral layer from the underlying ectoderm (Dziadek, 1979). Such a cell lineage scheme clearly differs from
one suggested earlier in which visceral and parietal endoderm were presumed to
share a common origin from a single pool of primitive endoderm cells (see
Gardner & Papaioannou, 1975; Gardner, 1978a, Fig. 1).
Cell lineage in mouse extraembryonic endoderm
111
The blastocyst injection experiments outlined earlier provided no evidence for
the existence in mature ICMs of cells capable of contributing to both visceral
endoderm and primitive ectodermal derivatives that one might expect according
to Dziadek's (1979) hypothesis (Fig. 1). However, they were uninformative
regarding the alternative possibility of a common origin for parietal and visceral
endoderm, because only the latter of the two extraembryonic endodermal layers
was analysed for chimaerism. Hence, more extensive cloning of 5th day 1CM
cells in blastocysts has been undertaken in which the resulting conceptuses were
recovered earlier in gestation than previously, thus enabling inclusion of the
parietal endoderm among the fractions analysed. These studies are reported in
the present paper, together with the results of additional experiments in which
the developmental potential of established parietal versus visceral endoderm
cells from post-implantation embryos was also examined by blastocyst injection.
A preliminary investigation had shown that visceral endoderm cells from both
embryonic and extraembryonic regions of 6th day egg cylinders could colonize
the visceral yolk sac endoderm of host conceptuses, albeit at a low frequency
(Rossant, Gardner & Alexandre, 1978). However, parietal endoderm was neither
used for injection nor included among the fractions analysed in these earlier
transplantations of post-implantation cells.
MATERIALS AND METHODS
Mice
Two stocks of mice were used, both of which had been derived from the PO
(Pathology Oxford) random-bred albino strain. The two stocks were homozygous for different alleles at the glucose phosphate isomerase (Gpi-1) locus
on chromosome 7, one being GPi-1*-/Gpi-1* and the other Gpi-lb/Gpi-lb
(Carter & Parr, 1967; De Lorenzo & Ruddle, 1969). The former was used to
provide host blastocysts and pseudopregnant recipients, and the latter donor
blastocysts and post-implantation embryos. All mice were maintained in controlled lighting and provided with food and water ad libitum. Most of the
Gpi-1&/Gpi-1& stock and all Gpi-lh/Gpi-lh mice were exposed to light between
07.00 and 19.00 h each day. Oestrous females were selected by vaginal inspection
(Champlin, Dorr & Gates, 1973) before onset of the dark period, paired with
fertile or vasectomized males, and inspected for vaginal plugs the following
morning. The day on which the plug was found was recorded as the first day of
pregnancy or pseudopregnancy. In addition, some matings were arranged
between Gpi-1&/Gpi-1& mice kept in a room which was in darkness between
14.00 and 23.00 h each day. These were used to provide more mature blastocysts that are assumed to be approximately 6 h ahead of those from females
mated in the more conventional lighting conditions. The former are referred
to hereafter as advanced and the latter as standard, host blastocysts.
178
R. L. GARDNER
Media
PB1 medium (Whittingham & Wales, 1969) was used for the recovery,
storage at room temperature and manipulation of embryos and, in addition, for
post-operative culture of the majority of injected blastocysts. It differed from
the original formulation in containing glucose (1 g/1) in place of lactate and
foetal calf serum (10 % v/v) in place of bovine serum albumin. Occasionally,
injected blastocysts were cultured in a-medium (Stanners, Eliceiri & Green,
1971) supplemented with 10 % (v/v) foetal calf serum because it supported more
rapid re-cavitation than PB1, and thus increased the chances of identifying the
location of the transplanted cells. One of two media (that of Whitten, 1971, in
earlier experiments, and that in Table 6.5 of Biggers, Whitten & Whittingham,
1971, in later experiments), from which calcium salts had been omitted, was
used for the culture of donor tissue between its exposure to proteolytic enzymes
and its final dissociation. Cultures were usually set up either in microdrops of
medium under liquid paraffin (Boots Pure Drug Co., U.K.) in 60x15 mm
plastic culture dishes (Falcon, Oxnard, U.S.A.) or in glass cavity cells, and
maintained at 37 °C in an appropriate gas phase. Injected blastocysts cultured
in PB1 were sometimes kept in hanging drops in manipulation chambers (Puliv,
Leitz, W. Germany) during incubation to facilitate detailed inspection during
recovery.
Recovery of embryos
Both standard and advanced host blastocysts were recovered between 15.45
and 18.00 h on the 4th and donor blastocysts between 13.00 and 15.45 h on the
5th day of pregnancy in all except one series of injections. The exception involved transplantation of earlier primitive ectoderm cells, for which advanced
host blastocysts were recovered between 12.15 and 12.45 h and donor blastocysts
between 09.15 and 09.45 h on the 4th and 5th day, respectively. The uterine
horns of 5th day pregnant females were distended with medium prior to
flushing, to aid release of the implanting blastocysts. Despite this measure, the
number of blastocysts obtained per female on the afternoon of the 5th day was
consistently lower than on the 4th day. Post-implantation donor embryos were
obtained by dissection, with watchmaker's forceps, of decidua from females
killed between 14.30 and 16.50 h on the 6th or 7th day of pregnancy.
Preparation of donor cells for blastocyst injection
Initially, ICMs were recovered microsurgically from 5th day blastocysts
(Gardner & Johnson, 1972), incubated for 10-15 min in 0-25% (w/v) pronase
(Calbiochem, Grade B) in Dulbecco ' A ' phosphate-buffered saline (PBS,
Oxoid, U.K.) at 37 °C, and then cultured in calcium-free medium for a further
30-45 min prior to dissociation. Since the yield of viable cells was rather
variable, the following protocol was adopted in the majority of experiments.
Cell lineage in mouse extraembryonic endoderm
179
The mural trophectoderm of donor blastocysts was first torn open with a pair
of siliconized (Repelcote, Hopkin & Williams, U.K.), sharp-tipped glass needles
controlled by Leitz micromanipulators. The opened blastocysts were then
exposed to a mixture of 0-25 % (w/v) trypsin and 2-5 % (w/v) pancreatin (both
from Difco Laboratories, U.S.A.) made up in calcium-magnesium-free Tyrode
saline (approximately pH 7-7) for 20-25 min at 4 °C. Thereafter, following a
brief rinse in PBl, the blastocysts were micromanipulated again with glass
needles in order to separate ICM tissue from investing trophectoderm and also,
in some cases, primitive ectoderm from primitive endoderm (Gardner, 1982).
The isolated ICMs or primitive ectoderms were incubated for 15 min in 0-25 %
pronase in PBS at 4 °C, rinsed in calcium-free medium, and then cultured for
up to 45 min in the latter at 37 °C. Finally, the 'loosened' cell masses were
transferred via a wide-bore pipette to a hanging drop of PBl in a manipulation
chamber, and dissociated by repeated aspiration through a siliconized pipette
whose tip had been heat polished down to an inside diameter of 30 fim or less
in a De Fonbrune microforge (Beaudouin, Paris). Virtually no cell death was
seen using this procedure, which yielded suspensions composed principally of
single cells and cell pairs.
For obtaining visceral endoderm cells, the embryonic or extraembryonic
regions of 6th and 7th day embryos were first isolated as described by Rossant
et al. (1978). They were then incubated in the trypsin-pancreatin mixture described above for approximately 15 min at 4 °C (Levak-Svajger, Svajger &
Skreb, 1969), followed by 0-25% pronase in PBS for a further 8 min at room
temperature. Finally, each fragment was rinsed and then cultured in calciumfree medium for 30-45 min before being restored to PBl and dissociated by
pipetting.
Difficulties were experienced in obtaining sufficient numbers of viable parietal
endoderm cells from 6th day embryos. Therefore, only 7th day embryos were
used as sources of these cells in the experiments reported in this paper. Intact
embryos were first cut in two with very fine iridectomy scissors (Weiss, U.K.)
below the level of insertion of Reichert's membrane and the egg cylinder held
by sucking its cut surface against a flame-polished pipette. Reichert's membrane
was then pulled off the egg cylinder by gripping the nipple-like extension at the
distal tip with watchmaker's forceps. Once isolated, Reichert's membrane was
incubated at 37 °C in pronase for 15 min followed by calcium-free medium for
up to 30 min, and then pipetted to release the parietal endoderm cells. It was
found in later experiments that enough viable cells were released spontaneously
if isolated membranes were incubated in trypsin and pancreatin at 37 °C for
30-40 min.
Injection of cells into blastocysts
The rough appearance of endoderm cells (Gardner & Rossant, 1979) was
used to distinguish them from ectoderm cells in disaggregates of 5th day ICMs
180
R. L. GARDNER
and of embryonic and extraembryonic fragments from post-implantation
embryos. Using a modified Leitz micromanipulator assembly, one or more cells
of a particular type were injected into each of a series of host blastocysts by a
technique that is described fully elsewhere (Gardner, 19786). Operated blastocysts were cultured for up to 90 min prior to inspection and then transplanted
to one or both uterine horns of females on the 3rd day of pseudopregnancy.
Analysis of conceptuses
Recipients were killed between 9 and 12 days after they had received injected
blastocysts, and conceptuses dissected as follows. First, Reichert's membrane
was dissected away from the edge of the chorioallantoic placenta, and trophoblast plus decidual tissue removed from its outer surface. The visceral yolk sac
was then separated from the base of the placenta, and the foetus, amnion and
umbilical cord removed from inside it. Next, the decidua basalts was separated
from the chorioallantoic placenta and discarded. The placenta, Reichert's
membrane and attached parietal endoderm cells, trophoblast (minus as much
decidua capsularis as possible), and foetus plus amnion and umbilical cord,
were handled as four separate fractions. They were rinsed in PBS, placed in
separate wells of a microtest plate (Nunc, Denmark), diluted with a small
volume of distilled water, and then frozen. Visceral yolk sacs were incubated in
the trypsin-pancreatin solution described earlier for a minimum of 3 | h at
4 °C, after which the endoderm was separated from the mesoderm in PB1. The
separated layers were then treated as described above for the other fractions.
In some cases, visceral yolk sacs and/or Reichert's membranes were cut in
two with iridectomy scissors prior to freezing so as to provide separate proximal
(placental) and distal (ab-placental) fractions for analysis. The technique for
separation and visualization of the glucose phosphate isomerase allozymes was
as described previously (Gardner, Papaioannou & Barton, 1973; Gardner &
Rossant, 1979), except that the 3 mm-thick starch gels were sliced in two following
electrophoresis, and the cut surface of each slice stained separately through
an agar overlay. Proportions of the two allozymes in a sample were estimated
visually from the electrophoretograms by two independent observers.
RESULTS
Transplantation of 5th day primitive ectoderm cells
Cells for injection were obtained either by dissociating microsurgically isolated
primitive ectoderm or, as previously, by selecting 'smooth' cells from disaggregated ICMs (Gardner & Rossant, 1979). A single cell was transplanted
into each blastocyst in one experiment, and either pairs or groups of up to six
cells, all or most of which were loosely attached to one another, in the remainder
(Tables 1 and 2). Donor cells typically adhered to the centre of the blastocoelic
surface of host ICMs, against which they were invariably injected. Standard
Stage of host
blastocysts
at injection
Code numbers
of chimaeras*
ca. 1400
1
Standard
SI-S3
4th day
16
Standard
20
18
15
S13-S27
ca. 1400
4th day
Standard
18
18
12
10
ca. 14-30f
S28-S37
4th day
4-6
10
Standard
ca. 13.00
S4-S9
4th day
Advanced
2, 3 or 4
15
21
15
12
S10-S12 + S38-S46
ca. 09-15
4th day
* These code numbers serve to identify the chimaeras individually in Table 2.
t All 18 blastocysts in this series of injections were transplanted to the oviducts of an immature female because no suitable pseudopregnant
recipients were available on the day of the experiment. The blastocysts were recovered 22£ h later and transferred to the uteri of females on the 3rd
day of pseudopregnancy. The lower than usual rate of normal development of these blastocysts may be due to this treatment.
Time of
recovery of
donor
blastocysts
No. of injected
blastocysts
transplanted
No. of
No. of cells to recipients
chimaeras
that became
Total no. of No. of normal among normal
injected/
pregnant
implantations conceptuses
blastocyst
conceptuses
Table 1. Rate of normal development and chimaerism following injection of 5th day primitive ectoderm cells into blastocysts
oo
3
§
*•»
182
R. L. GARDNER
Table 2. Contribution of donor cells in chimaeras produced by injecting 5th day
primitive ectoderm cells into blastocysts
si
Chimaera code no. . . .
PM
Stage of donor blastocysts*
I
No. of cells injected/blastocyst
Distribution + level of chimaerism
Parietal endoderm
Visceral yolk-sac endoderm
Visceral yolk-sac mesoderm
Foetus + amnion + umbilical cord
Placenta
Trophoblastic giant cells
Chimaera code no. . . .
Stage of donor blastocysts*
No. of cells injected/blastocyst
S3 S4
S5
S6
S7
S8
S9 SIO Sll SI2 S13lsi4 S15 S16 S17 S18 S!9 S20 S21
S22|S23
1 4 6 4-6 4-6 4-6
i I
14
IXN
S24 S25 S26 S27 S28 S29 S30 S31 S321 S33 S34 S35 S36 S37 S38 S39 S40 S41 S42 S43 S44 S45 S46
PM PM PM PM PM PM PM PM PM PM PM PM PM PM AM AM AM AM AM AM AM AM AM
Distribution + level of chimaerism
Parietal endoderm
Visceral yolk-sac endoderm
Visceral yolk-sac mesoderm
Foetus + amnion + umbilical cord!
I'liiccnin
lic
S2
PM PM PM PM PM PM PM PM AM AM AM PM PM PM PM PM PM PM PM PM PM PM
AVOW cvIK
i( • •
mmmwm
. : LLLI i I
._, I .......Li.....
I ' L i
Donor contribution: J , > 5 0 %
||||,ll-20%;
,41-50%;
]|f , 31-40%;
mil , 10% or less;
\~\ , none;
I
,21-30%;
f>7] , not analysed.
* PM, Donor embryos recovered on the afternoon of the 5th day.
AM, Donor embryos recovered between 09.10-09.40. on morning of 5th day of development.
t GPI activity in both the foetal fraction and visceral yolk-sac mesoderm of this chimaera was
exclusively of donor type.
rather than advanced 4th-day blastocysts were used as hosts in all experiments
except those involving transplantation of cells from blastocysts recovered on
the morning of the 5th day.
The rates of normal development and numbers of chimaeric conceptuses
obtained in the different series of injections are presented in Table 1. Table 2
provides details of the distribution and extent of contribution of the donor cells
in each of the 46 chimaeras that were obtained in these experiments.
A minor contribution of donor cells to the placental fraction was seen in
19 cases (Table 2), but is an uninformative observation because the embryonic
part of this organ is evidently composed of cells oftrophectodermal, primitive
ectodermal and primitive endodermal derivation (Duval, 1892; Snell & Stevens,
1966; Gardner & Papaioannou-, 1975). Hence, this fraction will not be considered
in the following appraisal of the distribution of donor cells.
Cell lineage in mouse extraembryonic endoderm
183
Forty-one of the chimaeras exhibited the B type of pattern of chimaerism
established earlier for primitive ectoderm cells (Gardner & Rossant, 1979) in
which donor cells were confined to the foetal fraction and/or visceral yolk-sac
mesoderm (all except S4, S29, S39, S40 and S41 in Table 2). Furthermore, the
donor contribution approached or exceeded 50 % in one or both of the fractions
in more than half these chimaeras. Indeed, donor allozyme activity alone was
detected in both the visceral yolk-sac mesoderm and foetal fractions of one
chimaera (S32, Table 2). Enzyme activity was absent from the visceral yolk-Sac
mesoderm of three conceptuses and too low for unequivocal scoring in a further
two. The reason for this is not clear, since no problem was encountered with the
corresponding endodermal fractions that had the same period of exposure to
trypsin and pancreatin.
Three of the five chimaeras that did not conform to the B pattern contained
donor cells in the trophoblastic fraction as well as in the foetal fraction and
visceral yolk-sac mesoderm (S4, S39 and S41 in Table 2). The extent of the
donor contribution to the trophoblast is almost certainly underestimated because of unavoidable dilution of this fraction with contaminating maternal
decidual cells. Donor cells were confined to the parietal and visceral yolk-sac
endoderm in one of the remaining two exceptional chimaeras (S29 in Table 2).
The other displayed a very minor contribution of donor cells to the parietal
endoderm fraction in addition to major contributions to the foetal and visceral
yolk-sac mesodermal fractions (S40). Since this last chimaera did not show
evidence of trophoblastic chimaerism, the parietal contribution cannot be
attributed to contamination of the latter fraction with cells from the former.
Allfiveexceptional chimaeras were produced in experiments in which more than
one cell was injected into each blastocyst. The distribution of chimaerism seen
in S4, S39, S40 and S41 (Table 2) may therefore be attributable to inadvertent
inclusion of one or more trophectoderm or primitive endoderm cells among
those transplanted rather than to lack of restriction in potency of primitive
ectoderm cells. The pattern observed in S29 is very difficult to explain, unless
one assumes that an attached group of primitive endoderm cells had been misclassified as primitive ectoderm cells. Notwithstanding, no case of chimaerism
specifically limited to the visceral yolk-sac endoderm, or to this tissue plus
the corresponding mesoderm and/or foetal fraction was encountered, even
among the 12 conceptuses colonized by donor cells recovered earlier on the 5th
day of development (S10-S12 and S38-S46 in Table 2).
Injection of 5th day primitive endoderm cells
Donor cells with a 'rough' appearance were selected, and injected either
centrally against the surface of the ICM of host blastocysts, or peripherally at
the junction between ICM and mural trophectoderm. Both advanced and
standard 4th day blastocysts were used as hosts, the former typically differing
from the latter in showing signs of endodermal differentiation and, in some
Total no.
of implantations
No. of
normal
conceptuses
Total no.
of chimaeras
among
normal
conceptuses
No.
chimaeric
in parietal
endoderm
only
t
No.
chimaeric
in visceral
endoderm
only
No.
chimaeric
in parietal
and
visceral
endoderm
No.
chimaeric
in
additional
or other
tissues
Code numbers
of chimaeras*
O
r
r
1
129
124
117
63
48
2
8+2?|
3J
R1-R48 + R68-R8O O
2 (attached)
25
23
22
19
16
0
3
0
R49-R67
w
* These code numbers serve to identify the chimaeras individually in Table 4.
t Donor allozyme was definitely present in the visceral endoderm fractions in these two chimaeras (nos. 29 and 30 in Table 4), but was equivocal in their
parietal endoderms.
% Donor allozyme was detected only in the foetal and placental fractions of one of these chimaeras (no. 2a in Table 4), and in the visceral mesoderm of the
second (no. 15a in Table 4). The third chimaera (no. 31 in Table 4) had a high proportion of donor allozyme in its visceral endoderm, plus a very weak contribution to the adjacent visceral mesoderm (see Fig. 2).
No. of cells
injected/
blastocyst
rNU. VI
injected
blastocysts
transferred
to recipients
that became
pregnant
Classification of chimaeras according to
distribution of donor cells
Table 3. Rate of normal development and chimaerism following injection of 5th day primitive endoderm cells into blastocysts
oo
185
Cell lineage in mouse extraembryonic endoderm
vc
PC
P
VC
V
PC
(B)
vc
PC
P
V
vc
(C)
V
(E)
P
VC
vc
V
M
MC
(F)
Fig. 2. Electrophoretograms of extraembryonic fractions of six of the single primitive
endoderm cell injection chimaeras presented in Table 4. A-E are examples of chimaeras in which the donor cell contributed to both visceral and parietal endoderm;
(A) = R15, (B) = R17, (C) = R26, (D) = R41 and(E) = R48. (F) shows the allozyme composition of the visceral endoderm and visceral mesoderm fractions in the
anomalous chimaera, R31. P, Vand M denote parietal endoderm, visceral endoderm
and visceral mesoderm fractions, respectively, and PC, VC and MC their corresponding mixed allozyme controls. Donor allozyme is represented by the upper,
more cathodally migrating band.
cases, loss of the zona pellucida as well. The majority of host blastocysts were
injected with single cells, the remainder each receiving an attached pair of cells
(Table 3).
Single cells
As shown in Table 3, 63 of the 117 normal conceptuses derived from bla$tocysts injected with single cells were found to be chimaeric, representing a cloning
efficiency of the donor cells of 54 %. The foetal fractions and/or visceral yolksac mesoderm were analysed in addition to both the parietal and visceral
yolk-sac endoderm in 50 of these chimaeras (nos. R1-R48, including R2A and
R15A, in Table 4). In all except three of the latter the progeny of the transplanted cell appeared to be confined exclusively to the extraembryonic endoderm. Two out of three of the exceptional chimaeras (nos. R2A and R15A in
Table 4) displayed patterns of colonization appropriate for primitive ectoderm
rather than primitive endoderm, and may therefore represent cases in which the
donor cell had been misclassified. The third (no. R31 in Table 4) exhibited a
high proportion of donor allozyme activity in the endoderm layer of the visceral
yolk sac, and a much lower proportion in the corresponding mesodermal layer
(Fig. 2 F). This conceptus was recovered on a day in which unusual difficulty
was experienced in separating the two layers of visceral yolk sacs following cold
enzyme treatment. The absence of donor allozyme activity from the foetal
fraction of this chimaera is consistent with the conclusion that its presence in
186
R. L. GARDNER
Table 4. Contribution of donor cells in chimaeras produced by injecting 5th day
primitive endoderm cells into blastocysts
R15
R7 R8 R9 RIO R l l R!2 RI3 R14 R15 A R16 R17 RI8 R19
R2 R2
A R3 R4 R5 R6
Rl
(A) Chimaera code no.
l
Donor cell(s) no. injected
Site of injection
c
Stage of host blastocyst
s
Distribution + level of chimaerism
Parietal endoderm
Visceral yolk-sac endoderm
Visceral yolk-sac mesoderm
Foetus + amnion + umbilical cord
Placenta
Trophoblast giant cells
1
1
1
1
1
1
1
1
1
C
C C
C
P
P
P
P
C
S
S
S
S
s
S
s
S
1
c
s s
1
1
1
1
1
c
s s s
C
C
C
C
S
S
S
S
1
1
1
C C
P
P
S
S
1
S
H- ....
— ..._ ... —
1
•
•
•
NA
X
x
III III
XX
1
C
1
1
xX X X X
xX XA XX
X X X X X X 'X
X
XXX X
R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 R40
'B) Chimaera code no.
...
1
1 1
1 1
1 1
1
1
1 1
1
1
1.
1 1
1
1
l
1
1
No. injected
Donor cell(s) site of injection
c C C C C C C C c C C C C C C C C C C C C
C C c C C C C C c 7 7
Location post-operatively*
c C C
s S S s s S S s s A A A A A S S s s s s S
Stage of host blastocyst
Distribution + level of chimaerism
Parietal endoderm
Visceral yolk-sac endoderm
Visceral yolk-sac mesoderm
Foetus + amnion + umbilical cord
Placenta
Trophoblast giant cells
xxxxxxx
xxxxxx
XXXXEx
(C) Chimaera code no. . . .
No. injected
Donor cell(s): site of injection
Location post-operatively*
Stage of host blastocyst
Distribution + level of chimaerism
Proximal parietal endoderm
Distal parietal endoderm
Proximal visceral yolk-sac endoderm
Entire visceral yolk-sac endoderm
Distal visceral yolk-sac endoderm
Visceral yolk-sac mesoderm
R41 R42 R43 R44 R45 R46 R47 R48 R49 R50 R51 R52 R53 R54 R55 R56 R57 RS8
(D) Chimaera code no. . . .
No. injected
Donor cell(s) site of injection
Location post-operatively*
Stage of host blastocyst
Distribution + level of chimaerism
Parietal endoderm
Visceral yolk-sac endoderm
R59 R60 R6I R62 R63 R64 R65 R66 R67 R68 R69 R70 R71 R72 R73 R74 R75 R76 R77 R78 R79 R80
Donor contribution: ^ , > 5 0 % ;
1111 , 10% or less;
NA, no GPI activity;
C, central
1
1
1
1
1
1
1
1
2
2
P
C
C
P
P
P
P
P
C
C C
P
?
?
P
P
P
P
P
7
7
A
A A A
A
A
A
A
A A
li
•
]
2
2
2
2
2
2
C C
C
C
C
C C
2
C c C C C P P
s s s s s s s s
C
• • 1JJJ m
III it
Ill Mi,,,,
!
III
III
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
C
C
C
C C
C
C
C
C
C
C
C
C
C
C C
C
C C
C
c
C
c c
c c
P
P
P
P
P
P
P
P
A
A A
A A
A A
A
A
A
A
A
A
A A A A
A
A A A
,41-50%;
Q
, none;
P, peripheral.
} | | , 31-40%;
^
A
!|| j | , 21-30%;
, fraction not analysed;
S, standard;
•Scored following re-cavitation of host blastocysts in short-term culture
A, advanced.
P
|jjj||, 11-20%;
Cell lineage in mouse extraembryonic endoderm
187
visceral mesoderm was due to contamination of the latter with visceral endoderm cells. None of the 28 chimaeras in which the trophoblast was analysed
exhibited colonization of this fraction (Table 4). A modest donor contribution
was evident in 2 out of 23 cases in which the chorioallantoic placenta was
examined. However, as noted earlier, these particular findings are not
instructive because of the composite nature of the embryonic part of this
organ.
Two further points are evident from Tables 3 and 4 concerning the single-cell
injections. First, in 8 of the 61 chimaeras in which the extraembryonic endoderm
had been colonized, progeny of the donor cell were detected unequivocally in
both its parietal and visceral layers (chimaeras R14, R15, R16, R17, R26, R41,
R48 and R74 in Table 4; and Fig. 2A-E). Equivocal results were obtained in
two further cases (R29 and R30 in Table 4) because of low enzyme activity in
the parietal endoderm. Secondly, chimaerism was confined to the parietal
endoderm in most cases, despite all but 12 conceptuses having developed from
blastocysts in which the donor cell had been placed against the centre of the
free surface of the ICM (Table 4). Indeed, only two chimaeras (or three if R31
is included) appeared to have donor cells restricted to the visceral yolk-$ac
endoderm. A marked bias is evident in the central injection into both standard
and advanced host blastocysts, 23 out of 29 in the former series (excluding R2A
and R15A, but including R31) and 16 out of 20 in the latter showing donor cells
in the parietal endoderm only. It is clear from Table 4 that, in some of the cases
in which the position of the donor cell could be established following postoperative culture, it had not remained at the site of injection (see R72-R80 in
Table 4). However, even when the cell did retain a central location (see chimaeras
R20-R22, R29-R37, and R68-R71, in Table 4), its progeny was found exclusively in the parietal endoderm in the majority of cases. Peripheral injection
of the donor cell -did not altogether preclude its making a contribution to the
visceral endoderm (see chimaeras R14, R41 and R48 in Table 4).
Attached pairs of cells
Pairs of 'rough' cells which had resisted separation during dissociation of
donor ICMs were selected for transplantation, and injected centrally into either
standard or advanced host blastocysts. As expected, the frequency of chimaerism
was higher than in the single-cell injections, and the donor contributions were
also usually greater (Tables 3 and 4). In the 10 chimaeras whose visceral yolksac mesoderm was analysed in addition to both parietal and visceral yolk*sac
endoderm, this fraction was devoid of donor allozyme activity. The distribution
of chimaerism with respect to parietal versus visceral yolk-sac endoderm once
again showed a strong bias towards the former fraction. Furthermore, the
10 chimaeras in this series in which distal and proximal halves of the extraembryonic endodermal layers were analysed separately (chimaeras R49-R58 in
Table 4) support the impression gained from the eight single-cell injection
188
R. L. GARDNER
chimaeras treated similarly (chimaeras R41-R48 in Table 4), of non-random
distribution of donor cells within the parietal endoderm itself. Thus, proximal
parietal endoderm was chimaeric in 14 and distal parietal endoderm in all
18 cases in which the two regions were analysed separately (Table 4C). In none
of the 14 in which both regions were chimaeric did the donor contribution to the
proximal obviously exceed that to the corresponding distal half. Contributions
were approximately equal in six conceptuses (R42, R44, R47, R48, R50 and
R54, Table 4), distal exceeding proximal in the remaining eight (R45, R51, R52,
R53, R55, R56, R57 and R58, Table 4).
Only two of the conceptuses whose proximal and distal visceral yolk-sac
endoderm regions were analysed separately exhibited chimaerism in this layer
(R48 in the single and R51 in the cell-pair injections, Table 4), and it was confined to the former region in both cases. Finally, visceral endodermal chimaerism
exceeded parietal in only 3 (R14, R48 and R63, Table 4) of the 11 conceptuses
obtained in both the single-cell and cell-pair injections in which both layers
were chimaeric. Levels of visceral and parietal chimaerism were approximately
equal in two cases (R17 and R74, Table 4), parietal exceeding visceral in the
remaining six (R15, R16, R26, R41, R51 and R66, Table 4).
Transplantation of 6th and 7th day endoderm cells
Several cells, often consisting of a mixture of both singletons and loosely
attached pairs of groups, were injected centrally against the blastocoelic surface
of the ICM of each host blastocyst. The origin and approximate numbers of
donor cells injected per blastocyst are indicated in Table 5, together with the
corresponding rates of normal development and chimaerism, and also the
frequencies of different patterns of colonization. Estimates of the extent to
which donor cells contributed to the fractions that they colonized are presented
in Table 6 for all 34 chimaeras obtained in these experiments. Three fractions,
parietal endoderm, visceral yolk-sac endoderm and visceral yolk-sac mesoderm,
were analysed in all but seven conceptuses. Additional fractions were analysed
in a proportion of conceptuses developed from blastocysts which had received
parietal or visceral endoderm cells from 7th day donor embryos (see Table 6 for
details). In no case were donor cells detected in fractions other than the parietal
and/or visceral endoderm.
A high frequency of chimaeras was obtained with 6th day visceral endoderm
cells, regardless of whether they were isolated from the embryonic or extraembryonic region of the egg cylinder. In addition, donor cells showed as strong
a bias towards parietal rather than visceral endodermal colonization as endoderm cells from 5th day donor embryos (cf. Tables 3 and 5, and Fig. 3). The
rate of chimaerism fell dramatically when 7th day visceral endoderm cells were
transplanted (6 % overall as opposed to 79 % for 6th day cell injections).
Nevertheless, three of the four chimaeras showed exclusively parietal endodermal
colonization, donor cells being detected in the visceral endoderm layer only in
6th day visceral
embryonic endoderm
6th day visceral extraembryonic endoderm
7th day visceral
embryonic endoderm
7th day visceral
embryonic/extraembryonic junctional
endoderm
7th day visceral extraembryonic endoderm
7th day parietal
endoderm
Origin of donor cells
2
4 + 1?
0
2
4+1?
8
17
25
22
40
4-5
5-6
3-10
0
1
0
0
0
0
0
0
0
P1-P4
V29 + V30
V27 + V28
V24-V26
0
0
1
V1-V23
Code numbers
of chimaeras*
1
No.
chimaeric
in parietal
and
visceral
endoderm
3
No.
chimaeric
in visceral
endoderm
only
* These code numbers serve to identify the chimaeras individually in Table 6.
31
2
3
40
3
61
3
ca.5
19
5
23
4-5
30
No. of
normal
conceptuses
32
No. of
blastocysts
injected
No.
chimaeric
in parietal
endoderm
only
ca. 5
No. of cells
injected/
blastocyst
No. of
chimaeras
among
normal
conceptuses
Classification of chimaeras according to
distribution of donor cells
I
yonic en
00
SO
dpop
Table 5. Rate of normal development and chimaerism following injection of endoderm cells from post-implantation embryos into blastocysts
lineage in mouse extra
190
R. L. GARDNER
Table 6. Contribution of donor cells in chimaeras produced by injecting visceral
or parietal endoderm cells into blastocysts
Chimaera code no. . . .
Source of donor cells*
Age of donor embryosf
No. cells injected/blastocyst
Distribution + level of chimaerism
Parietal endoderm
Visceral yolk-sac endoderm
Visceral yolk-sac mesoderm
Foetus + amnion + umbilical cord
Placenta
Chimaera code no. . . .
Source of donor cells*
Age of donor embryosf
No. of cells injected/blastocyst
VI
V5
V6 V7
V8 V9 V10 Vll V12 VI3 V14 V15 V16 VI7
Em Em Em Em Em
Em Em
Em Em Em Em Em Em Em Km Em Em
6
Jlj), 10% or less;
6
V3 V4
6
6
6
6
6
6
6
6
6
6
6
6
4 5
6
6
6
4-5
5
5
xxxxxxx
xxxxx
PI
ei
P3
P4
Ex
Ex
P
p
P
P
7
7
7
7
7
7
V18 V19 V20 V21 V22 V23 V24 V25 V26 V27 V28 V29 V30
Em Em Em Em Em Em
6
6
6
6
6
6
5
5
5
5
5
5
Distribution + level of chimaerism
Parietal endoderm
Visceral yolk-sac endoderm
Visceral yolk-sac mesoderm
Foetus + amnion + umbilical cord
Placenta
Trophoblastic giant cells
Donor contribution:
V2
| | | , 21-30%;
Ex
Ex
Ex
6
6
6
4-5 4-5 4-5
Em Em
7
7
5
5
5-6 5 - 6 5-6
8-10 8-10 6
xxxx
xxxx
XXX
||j,j , 11-20%;
[_],none; [><j, not analysed.
* Em, Visceral endoderm from embryonic region of egg clinder,
Ex, Visceral endoderm from extra-embryonic region
t 6 = 6th day and 7 = 7th day embryos
the fourth chimaera (V27-V30 in Table 6 and Fig. 3). The somewhat higher rate
of chimaerism obtained in the 7th day parietal endoderm cell injections (13 %)
is probably attributable to the fact that, in general, more cells were transplanted
into each blastocyst than was the case in the corresponding visceral endoderm
injections (Table 5). Colonization of their tissue of origin only was seen in all
four unequivocal chimaeras produced in parietal endoderm cell injections
(P1-P4 in Table 6).
Two further points are worthy of note. First, donor contributions are somewhat variable but, as is evident from comparison of Tables 4 and 6, generally
lower than those obtained by injection of single cells or cell pairs from 5th day
donors. Secondly, parietal endoderm fractions that had been colonized by postimplantation visceral endoderm cells appeared indistinguishable from those that
had not when inspected at a magnification of x 50 during removal of contaminating trophoblasts from Reichert's membrane.
Cell lineage in mouse extraembryonic endoderm
C
V1
V20 C
V2
V3
V4
V5
V6
V24 C
V7
V8
V9
191
V10
V29 C
Fig. 3. Electrophoretograms of the parietal endoderm fractions of chimaeras VI,
V10, V20, V24 and V29 in Table 6, obtained by injection into blastocysts of visceral
endoderm cells from post-implantation embryos. Seventh day embryos were used to
provide donor cells in the case of V29, and 6th day embryos in the remainder;
Control samples are denoted by C. Donor allozyme is represented by the upper, more
cathodally migrating band.
DISCUSSION
5th day ICM cell injections
Altogether, 128 chimaeras were obtained in these experiments. All except 5
could be assigned unequivocally to one of two classes on the basis of the distribution of chimaerism. Thus, donor cells were confined to the extraembryonic
endoderm only in 80 chimaeras, and to the extraembryonic mesoderm of the
visceral sac and/or foetal fraction in a further 43. Furthermore, the pattern of
chimaerism corresponded with the morphological type of donor cells injected
in all but 3 of these 123 chimaeras. In one of the three exceptions, putative
primitive ectoderm cells colonized the extraembryonic endoderm (chimaera S29
in Table 2), while in the other two putative primitive endodermal cells colonized
the foetal or visceral yolk-sac mesoderm fractions (chimaeras R2A and R15A
in Table 4). These presumably represent instances in which donor cells were
misclassified, possibly because, as found earlier, the morphological distinction
between the two categories of ICM cell depends rather critically on their treatment during and after dissociation (Gardner & Rossant, 1979).
Four of the remaining five chimaeras that did not conform to either of the
above patterns of colonization were obtained in experiments in which several
cells were injected into each host blastocyst. Hence, they may represent cases in
which a mixture of primitive ectoderm plus polar trophectoderm (S4, S39, and
S41, Table 2) or primitive endoderm cells (S40, Table 2) were transplanted
inadvertently. Contamination of donor ICMs or primitive ectoderms with polar
trophectoderm cells might well occur if the latter tissue had already embarked
on the process of thickening that leads to extraembryonic ectoderm formation
7-2
192
R. L. GARDNER
(Copp, 1979). Extraembryonic ectoderm cells resemble those of the primitive
ectoderm in their smooth appearance, and can clearly colonize the trophectodermal derivatives of host embryos following blastocyst injection (Rossant, et al.
1978). Likewise, the distinction between primitive ectoderm and endoderm cells
is less marked in earlier 5th day blastocysts which were used as donors in the
experiments in which S40 was obtained. The fifth anomalous chimaera, in which
donor cells were detected in both endoderm and mesoderm layers of the visceral
yolk sac, was produced in a series of experiments in which a single cell was
injected into each host blastocyst (R31 in Table 4). This is the only instance in
which a 5th day ICM cell clone appeared not to partition between the extraembryonic endoderm and other ICM derivatives. However, as noted earlier,
there are grounds for suspecting that contamination of visceral yolk-sac mesoderm with corresponding endodermal cells may account for this particular
result.
If the two extraembryonic endoderm layers had separate origins, as proposed
by Dziadek (1979), individual early endoderm cells would be expected to
colonize either the parietal or the visceral endoderm of host embryos (Fig. 1).
In fact, at least 13% of clones clearly spanned both tissues. In addition, one
might expect to find primitive ectodermal clones that contributed specifically to
the visceral layer of the extraembryonic endoderm as well as to the corresponding mesodermal layer and/or foetus (Fig. 1). No unequivocal example of such
clones was found. One explanation for the failure to detect them might be that
they originate from morphologically intermediate ICM cells (Gardner &
Rossant, 1979) whose transplantation was avoided in the present experiments.
This is most improbable because, although intermediate cells are usually found
in both isolated entire primitive ectoderms and corresponding endoderms
(Gardner & Rossant, 1979), these two tissues consistently yield identical
patterns of chimaerism to dissociated 'smooth' and 'rough' cells, respectively,
following injection into blastocysts (Gardner, 1982). A further possibility, that
the cells in question are no longer present at the stage at which donor embryos
were recovered, is equally unlikely. Although individual earlier ICM cells can
indeed contribute to both extraembryonic endoderm and other ICM derivatives
of host embryos, they do so to the parietal as well as the visceral layer as often
as to the latter alone (Rossant & Gardner, 1982).
One further point about Dziadek's hypothesis concerns the in vitro experiments on which it is based. Regeneration of endoderm has been observed consistently only in ICMs isolated from giant blastocysts composed of three or
more embryos (Pedersen et al. 1977; Dziadek, 1979). Similar investigations on
ICMs from standard blastocysts have yielded completely negative results
(Sherman, Strickland & Reich, 1976; Hogan & Tilly, 1977; D. Solter, personal
communication of unpublished observations) or, at best, a very modest rate of
regeneration (Atienza-Samols & Sherman, 1979). Even when regeneration is
obtained the evidence attributing it to primitive ectoderm cells is not com-
Cell lineage in mouse extraembryonic endoderm
193
pelling (Gardner, 1981). Indeed, in very recent experiments it has been found
that, whereas 'primitive ectoderms' isolated by immunosurgery from 5th tfay
blastocysts almost invariably regenerated an endodermal layer, those isolated
microsurgically did not do so even if their cell number was enhanced by aggregation (Gardner, 1982). The reason for this discrepancy seems to be that the
endoderm is already multilayered by the stage at which it is clearly discernible,
so that some of its cells are protected from exposure to the immunosurgical
reagents. Results of both in vitro and in vivo experiments lead to the conclusion
that it is from these residual endoderm cells rather than the primitive ectoderm
that the entire regenerated layer is derived (Gardner, 1982).
The fact that single 5th day endodermal cell clones can contribute to both
the parietal and visceral endoderm of host embryos is clearly consistent with
the alternative hypothesis that the two types of extraembryonic endoderm cell
share a common precursor (Gardner & Papaioannou, 1975; Gardner, 1978a).
Post-implantation endoderm cell injections
Donor cells were detected exclusively in the extraembryonic endoderm in all
chimaeras produced in these experiments, despite the screening of one or more
additional fractions in each case. Sixth day visceral endoderm cells gave a
relatively high rate of chimaerism, and displayed as strong a bias towards
parietal rather than visceral colonization as 5th day endoderm cells. Nevertheless, the level of chimaerism was usually lower, suggesting that 6th day cells
form smaller clones following transplantation than 5th day cells. Seventh day
visceral endoderm cells yielded a very low rate of chimaerism compared with
their 6th day counterparts, regardless of whether they came from the embryonic
part of the egg cylinder, the extraembryonic part, or from the junctional jzone
between the two. However, since the donor contributions in the four chimaeras
compared favourably with those produced by similar numbers of 6th day cells,
the difference between the two stages may be attributable to a lower frequency
of clonable cells in 7th day visceral endoderm rather than to a reduction in size
of clones that such cells can form.
What is the significance of the unexpected finding that established visceral
endoderm cells yielded exclusively parietal chimaerism following injection into
blastocysts in the vast majority of cases? Do the transplanted cells actually
undergo phenotypic change, or do they retain their visceral characteristics and
simply accumulate in the parietal endoderm through failure to become integrated in their normal site? The latter seems most unlikely, especially since, even
in chimaeras in which donor allozyme accounted for 20% or more of the total
glucose phosphate isomerase activity, no morphological peculiarities could be
discerned in this tissue and post-implantation development appeared to have
proceeded normally. Support for the former possibility can be found in other
studies. Thus, Diwan & Stevens (1976) obtained histological evidence that
visceral endoderm tissue isolated from 6th day mouse embryos can form
194
R. L. GARDNER
parietal endoderm when transplanted to the testis. In addition, Hogan & Tilly
(1981) have provided both ultrastructural and biochemical evidence that transformation of visceral into parietal endoderm cells can also take place in 7th day
egg cylinders in vitro. However, it is not clear either in these studies or the
present one, whether all visceral endoderm cells retain this option, or only a
subpopulation of relatively immature stem cells. The dramatic drop in frequency
of chimaeras when 7th as opposed to 6th day visceral cells were injected into
blastocysts is certainly consistent with the latter possibility.
Four chimaeras were produced by transplanting 7th day parietal endoderm
cells, but despite injection of up to 10 cells per blastocyst the donor contribution
was very modest in each case. Given the extent to which parietal chimaerism is
favoured even with visceral endoderm, a much larger series of chimaeras would
have to be analysed in order to establish whether parietal cells retain the option
of forming their visceral counterparts. Furthermore, the significance of any
positive results would be difficult to evaluate unless higher donor cell contributions were achieved than in current injections using these cells.
General considerations
The much higher rate of parietal than visceral chimaerism that is evident in
the present endoderm cell injections affords an explanation for the relative
paucity of chimaeras in those undertaken earlier in which only the visceral
component of the extraembryonic endoderm was analysed (Rossant et ah 1978;
Gardner & Rossant, 1979). This very marked bias towards parietal chimaerism
does not seem to depend simply on preferential attachment of donor cells to
the mural trophectoderm rather than the ICM of host blastocysts. Some 5th
day endoderm cells that were placed on the ICM clearly shifted peripherally
during post-operative culture. However, a majority of'parietal endoderm only'
chimaeras was obtained even in those cases in which the donor cells remained
attached to the ICM (Table 4). In addition, the distribution of chimaerism did
not seem to be influenced by the stage of host blastocysts at injection, relatively
earlier blastocysts giving similar results to the more advanced ones which already
showed signs of endodermal differentiation. A further possibility is that donor
endoderm cells that colonize the visceral layer may be less likely to survive if,
for example, the visceral embryonic endoderm undergoes degeneration rather
than displacement during formation of the definitive embryonic endoderm
(Gardner, 1978 a). However, this would not account for the finding that, even
within the parietal endoderm, distal chimaerism appears to be favoured over
proximal. It also fails to explain the fact that those single ICM cells from early
4th day blastocysts which yield extraembryonic endodermal chimaerism following blastocyst injection do not show preferential parietal colonization.
The majority of clones formed by such cells contribute to both tissues of the
extraembryonic endoderm, those confined to the parietal layer being no more
Cell lineage in mouse extraembryonic endoderm
195
common than those confined wholly to the visceral yolk-sac endoderm (Rossant
& Gardner, 1982).
The work of Hogan & Tilly (1981) suggests that disruption of the epithelial
organization or alteration in the cellular substratum of visceral endoderm cells
may be responsible for their conversion to parietal endoderm cells. It raises the
interesting possibility that such factors may also play a role in the normal
differentiation of the extraembryonic endoderm. Thus, initially, all primitive
endoderm cells are in contact with primitive ectoderm cells because they
evidently differentiate as a monolayer on the blastocoelic surface of the ICM.
However, their increase in number in the late blastocyst is not accompanied by
a corresponding increase in surface area of the underlying ectoderm. Hence,
mitoses in the primitive endoderm are likely to be so oriented that, in general,
only one daughter cell remains adjacent to the primitive ectoderm, the other
being forced into a new superficial layer (Fig. 4). In addition, peripheral endoderm cells may suffer direct lateral displacement on to the adjacent mural
trophectoderm. Release from contact with the ectoderm by either means might
be the critical event enabling the cells to migrate peripherally and differentiate
into parietal endoderm. Once implantation has occurred, rapid expansion of the
ectodermal surface during egg-cylinder formation would permit both daughters
of dividing visceral endoderm cells to remain within this layer (Fig. 4). This
scheme accounts for the multi-layering of endoderm cells seen in the blastocyst
prior to parietal endoderm formation (Gardner, 1982). It also explains the
paucity of visceral relative to parietal endoderm cells in the late blastocyst, and
the rapid increase in number of the former following implantation (Enders,
Given & Schlafke, 1978).
The blastocyst injection data may be explained as follows. Single early ICM
cells that undergo endodermal differentiation following transplantation typically
contribute to both parietal and visceral layers because they are efficiently
incorporated into the primitive endoderm cell monolayer in host blastocysts
(Fig. 4). Differentiated endoderm cells, by contrast, are less likely to achieve
the necessary integration following isolation, and will therefore tend to form
the first and hence most distally migrating parietal endoderm cells (Fig. 4).
Available data on early differentiation of the extraembryonic endoderm in
the rodent embryo can thus be accommodated in the above scheme. However,
many questions need to be answered before the underlying processes can be
understood in detail. For example, is transformation to parietal cells a property
of all visceral cells or of a specific subpopulation within this layer that retains
a primitive endodermal character? Is it an irreversible or reversible change?
How late in development can it occur, and does it play a role in normal growth
of the parietal yolk sac? Recent studies on endodermal differentiation in embryonal carcinoma cells in culture in response to defined molecules offer a promising
system for examining more closely the relationship between parietal and visceral
cells (e.g. Hogan, Taylor & Adamson, 1981; Strickland, & Mahdavi, 1978;
196
R. L. GARDNER
Fig. 4. Diagram illustrating the scheme proposed for initial steps in differentiation
of the extraembryonic endoderm. It is assumed that endoderm cells must remain
adjacent to either primitive ectoderm or diploid extraembryonic ectoderm tissue
(Hogan & Tilly, 1981) in order to express the visceral phenotype. (A) represents a
blastocyst at the end of the fourth day of development in which the primitive endoderm has recently delaminated. One cell in this layer, whose subsequent development
is followed in B-D, is depicted in black. The area of contact between the endoderm
and underlying primitive ectoderm remains more or less constant until the blastocyst phase of development is completed. Hence, when the cell divides in the later
blastocyst (B), only one daughter can retain the position of the parent cell. The
other daughter is therefore released into a new superficial layer where it is free to
migrate laterally and contribute all its progeny to the parietal endoderm (C). Subsequent division of the daughter remaining adjacent to the ectoderm may be oriented
in one of two directions, depending on whether it occurs prior to or during expansion
of the ectodermal surface. In the former case, it will repeat the pattern exhibited by
the parent cell (B). In the latter, both daughters may remain adjacent to the ectoderm (C). The end result is that individual primitive endoderm cells would normally
be expected to contribute mitotic descendants to both the parietal and visceral endoderm in the post-implantation embryo (D). Transplanted early ICM cells typically
behave thus, when they contribute to the extraembryonic endoderm, presumably because they can readily establish appropriate relations with primitive ectoderm cells
in host blastocysts. Transplanted primitive and visceral endoderm cells, on the other
hand, are presumed to yield preferential parietal colonization because, once disrupted, their contacts with the ectoderm are not readily re-established. Cell outlines
are indicated only for primitive endoderm and its parietal and visceral derivatives.
Trophectoderm and its derivatives are stippled, while the primitive ectoderm has been
left blank.
Cell lineage in mouse extraembryonic endoderm
197
Strickland, Smith & Marotti, 1980). Nevertheless, much more needs to be
learned about the patterns of growth of parietal and visceral endoderm in the
embryo and, in particular, the properties of cells in the region of continuity
between the two layers of extraembryonic endoderm that is eventually incorporated into the placenta.
I wish to thank Dr R. Beddington, Mr S. Buckingham, Mrs W. Gardner, Dr C. F. Graham,
Professor H. Harris, Mr K. Mabbatt and Mrs J. Williamson for help in preparation of the
manuscript, and Mrs M. Carey and Mrs L. Ofer for technical assistance. The work was
supported by the Medical Research Council and the Royal Society.
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inner cell mass of the mouse blastocyst: effects of conditioned medium. /. exp. Zool. 208,
67-71.
BIGGERS, J. D., WHITTEN, W. K. & WHITTINGHAM, D. G. (1971). Culture of mouse embryos
in vitro. In Methods in Mammalian Embryology (ed. J. C. Daniel), pp. 86-116. San
Francisco: Freeman.
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(Received 15 October 1981)