/. Enibryol exp. Morph. Vol. 60, pp. 419^28, 1980
419
Printed in Great Britain © Company of Biologists Limited 1980
In vitro development of inner cell masses isolated
from fjf and tw5/tw5 mouse embryos
By BRIGID HOGAN, 1 MARTHA** SPIEGELMAN 2
AND DOROTHEA BENNETT 2
From the Imperial Cancer Research Fund, Mill Hill Laboratories, London
SUMMARY
Inner cell masses (ICMs) isolated immunosurgically from mouse blastocysts segregating
the homozygous lethal mutants t°/t° and rw5/'w5 were cultured in vitro. Presumed t°/t° ICMs
fail to grow after three days in culture (equivalent gestational day 7-5) when they consist of
an outer layer of endoderm cells surrounding about 30 epiblast cells. Presumed homozygous
r v5 /r v5 ICMs develop to a more advanced stage in culture and on the seventh day (equivalent
gestational day 11-5) consist of an inner core of disorganized ectoderm cells with a small
proamniotic cavity, surrounded by multiple layers of endoderm cells.
INTRODUCTION
Mouse embryos homozygous for the mutations t° (or its allele, t6) and Vv5 die
during the early post-implantation period (Gluecksohn-Schoenheimer, 1940;
Bennett & Dunn, 1958) but the mechanism behind the lethality of these and
other mutations within the T/t complex is unknown (for reviews see Bennett,
1975; Sherman & Wudl, 1977). The discrete morphological abnormalities seen
in the different classes of mutant homozygotes suggested that they have defects
in cell-cell interaction, raising the possibility that T/t genes code for cell
surface components specifically required during embryogenesis (Bennett, 1975).
Indeed, there is some serological evidence that T/t related antigens are present
on pre-implantation mouse embryos (Artzt, Bennett & Jacob, 1974; Kemler
et al. 1976). Alternatively, it has been argued that the primary effect of t
mutations is on the intermediary metabolism of all cells in the embryo, leading
to the death of the embryo at certain stages of development (Nadijka &
Hillman, 1975; Wudl, Sherman & Hillman, 1977).
One way to distinguish between these hypotheses is to transplant embryos
in vivo to ectopic sites. According to the ' organisational defect', hypothesis one
might expect cells of the mutant embryos to be capable of continued growth
under these conditions, and this appears to be the case with homozygous T
1
Author's address: Imperial Cancer Research Fund, Mill Hill Laboratories, Burtonhole
Lane, London, NW7 IAD, U.K.
3
Authors'1 address: Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New
York, 10021, U.S.A.
420
B. HOGAN, M. SPIEGELMAN AND D. BENNETT
embryos (Ephrussi, 1935; Bennett, Artzt, Magnuson & Spiegelman, 1977). On
the other hand, homozygous t6 and t° embryos fail to grow in ectopic sites
(Wudl et al. 1977; Artzt, 1978 personal communication).
Another approach is to culture embryos segregating homozygous t mutants
in vitro. Using this technique two groups (Erikson & Pederson, 1975; Wudl &
Sherman, 1976; Wudl et al. 1977) have reported that cells of presumed
homozygous t*/t6 and ?Av5/?w5 embryos fail to grow in culture beyond the 7th
or 11th equivalent gestational day respectively. In these experiments intact
blastocysts were allowed to attach to the surface of the culture dish and the
inner cell mass (ICM) remained in contact with the outgrowing sheet of
trophectoderm cells. Under these conditions, interactions between trophectoderm and ICM affecting cell survival are still possible. Recently, Wudl and.
Sherman (1978; Wudl et al. 1977) have reported abnormalities in the growth
of presumed t6/t& trophectoderm cells when they are cultured in the absence of
the ICM. In this paper we have investigated the in vitro development of both
t°/t° and tw5/t"'5 ICMs when they are isolated from the trophectoderm by the
technique of immunosurgery. Normal ICMs isolated in this way can continue
to develop in culture into structures resembling the egg cylinder of normal
embryos at around 7-5 days of gestation (Wiley, Spindle & Pederson, 1978;
Hogan& Tilly, 1978 a).
MATERIALS AND METHODS
Mice
+ /t° and + // w 5 males and females were obtained by crossing T/t° and
7/fw5 males with + / + random bred CFi females. The F 2 hybrid males were
progeny tested by mating to T/+ females, and their relative transmission of
t to + measured by the ratio of tailless (T/t) to Brachyury (77 + ) offspring;
this ratio was unexpectedly high for the males used in these experiments. For
the +/t° males the ratios were 20:0, 45:2, 46:3, 37:3, 9:2, 22:3, and 29:4,
giving an average transmission frequency of 92-4%. For the +/fw6 males the
ratios were 12:0 and 11:0 giving a transmission frequency of 100%.
To obtain homozygous mutant embryos, +/t F± hybrid females were superovulated and mated with + /t males. For control crosses, either the Brachyury
77 + litter-mates of the + ft females, or + / + CFX females, were mated with
+ jt males.
Fig. 1. Phase contrast microscopy of ICMs after three days in culture. ICMs were
isolated immunosurgically from 26 blastocysts of either experimental (+/f°x +/t°)
or control (T/ + x + ft0) crosses and cultured in parallel under the same conditions.
(a) 25/26 ICMs from the experimental cross. The ICMs classified as presumed
t°/t° are labelled with arrows; some of these have remnants of dead trophoblast
still attached.
(b) 22/26 ICMs from the control cross; in most of them the inner ectoderm has
expanded into a hollow vesicle. Scale bars = 100 /im.
Cultured ICMs from t mutant mouse embryos
K
i ^ ! ^
•**.
>$-*H
\-j»\:i
&£ '„
4
.
421
422
B. HOGAN, M. SPIEGELMAN AND D. BENNETT
Embryos
Blastocysts were collected on the fourth day of pregnancy when the day on
which the vaginal plug is observed is day 1. The zonae pellucidae were removed
with acidic tyrode solution, pH 2-5, and the blastocysts incubated overnight in
Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine
serum. Inner cell masses were isolated immunosurgically the following day
(equivalent to 4-5 days p.c), and cultured as described by Hogan & Tilly
(1978 a, b). The cultures were observed daily and photographed with an inverted
phase-contrast microscope.
Inner cell masses were fixed overnight in 2 % glutaraldehyde in 0- 1M phosphate
buffer, post-fixed in osmium tetroxide, dehydrated through acetone, and
embedded in Epon. Serial sections from 1 to 2 /<M in thickness were stained
with 1 % toluidine blue in 1 % borax solution (pH approximately 11), photographed, and the number of interphase nuclei and mitotic cells counted from
serial reconstruction of the ICMs.
RESULTS
Inner cell masses from t°/t° embryos
After one day in culture (equivalent gestation day e.g.d. 5-5) there is no
significant difference in the size or morphology of the inner cell masses isolated
from experimental ( + /f°x +/t°) or control (T/+ x +/t°) blastocysts. After
two days, all the ICMs have an outer layer of endoderm surrounding an inner
core of epiblast cells but some of the ICMs in experimental cultures are
significantly smaller than the controls. By day three most of the control ICMs
develop a central cavity and begin to expand (Fig. 1 b), but only about half of
the ICMs in experimental cultures behave in this way (Fig. 1 a). The remainder
fail to increase in size and gradually degenerate over the next few days. As
shown in Table 1, of 344 ICMs isolated from experimental blastocysts, 50%
died by the seventh day of culture (e.g.d. 11-5). This is close to the expected
proportion of homozygous mutant embryos in the cross +/t°x + /t° (approx.
46%) and suggests that the ICMs which fail to increase in size have the
genotype t°/t°.
The morphology of typical presumed t°/t° and normal (+ / + or + /t°) ICMs
fixed on day three of culture is shown in Fig. 2 a and b. The epiblast cells of
the presumed mutants are rounded and disorganized and do not develop the
equivalent of a proamniotic cavity. The average number of epiblast cells in
eight presumed t°/t° ICMs fixed on day 3 of culture and serially sectioned was
30 (range 22-45). In some of these ICMs a high percentage of the cells were
in mitosis. (This prompted us to determine the percentage of cells in mitosis for
eight ICMs and we obtained the values 4, 7, 7, 11, 12, 13, 13 and 36%.) Many
of the endoderm cells of the presumed t°/t° ICMs are highly vacuolated.
Cultured ICMs from t mutant mouse embryos
423
Table 1. Survival of ICMs in vitro
ICMs were isolated immunosurgically from blastocysts and cultured in vitro
for 7 days as described in Materials and Methods. Since it was observed that
presumed t°/t°ICMs degenerate after day 3, the percentage of homozygoas mutants
in the cross (+ /t° x + //°) was estimated from the difference between the number of
ICMs put into culture and the number surviving on day 7. In the experimental cross
(+// w 5 x +// w 5 ), the percentage of ICMs with a distinctly abnormal morphology
(see Fig. 3 a) was scored on day 7 of culture. Each row represents one experiment in
which ICMs were isolated from blastocysts collected from 1-5 mice on the same day
and cultured together in the same dish.
Crosses
ICMs
surviving for
7 days in
culture
No. of ICMs
(%)
Presumed
t°/t°
(%)
Experimental
+ /t°x +/t°
Total
57
49
52
31
42
53
54
26
46
99
56
78
344 Average 50 Average
51
48
58
46
54
44
50
Control
100
96
100
94
—
—
—
—
Total 103 Average 98
—
+ /t°xT/ +
T/+x+/t°
Crosses
35
25
26
17
ICMs
ICMs on
surviving for day 7 with
abnormal
7 days in
morphology
culture
No. of ICMs
(%)
(%)
Experimental
+ // w 5 x +// w 5
Total
Control
T/+ x +/t™5
+ /+X +/P' 5
Total
32
50
84
6
100
50
45
75
49
65
95
31
166 Average 86 Average 37
14
100
68
88
82 Average 94
424
B. HOGAN, M. SPIEGELMAN AND D. BENNETT
(a)
^i> v>.;j
•
-
*
,
.
•
_
.
.
» , . « *
Fig. 2. Sectioned ICMs on day 3 of culture. ICMs were prepared from blastocysts
of the cross (+//°x +//°) and cultured in vitro for three days, (a) Presumed /°/'°
ICM showing outer endoderm cells and an inner core of epiblast cells, three of
which are in mitosis, (b) Presumed normal (+ /t° or + / + ) ICM with the embryonic
ectoderm surrounding a small proamniotic cavity. Scale bars = .10/im.
Cultured ICMs from t mutant mouse embryos
5 (a)
425
"
3(b)
Fig. 3. Presumed ^w5//w6 ICMs after 7 days in culture, (a) Phase-contrast microscopy
of two presumed homozygous mutant ICMs showing proliferation of rounded
endoderm cells on the outside and small, densely filled inner cavities. The presumed
mutant ICMs are attached to a third, normal expanded ICM, Scale bar = 50/*m.
(b) Section of a presumed tw5//w8 ICM showing the accumulation of extracellular
material, presumably basement membrane material (arrow), between the outer
endoderm cells and the inner ectoderm cells. Scale bar = 20 /tm. (c) A presumed
normal ( + / + or + /fw5) ICM showing the outer epithelial layer of endoderm cells
separated by spaces from the inner ectoderm cells which enclose an expanded cavity.
Scale bar = 40 /im.
426
B. HOGAN, M. SPIEGELMAN AND D. BENNETT
However, similar vacuolated cells are seen in the presumed + / + or +/t°
ICMs (Fig. 2b), as well as in cultured ICMs from control crosses and normal
C3H embryos (unpublished observations; Hogan & Tilly, 1978a).
Inner cell masses from t w5 /t w5 embryos
For the first three days in culture there is no significant difference between
the development of ICMs from control (T/+ or + / + x + /r v 5 ) and experimental ( + /f w5 x +/7 w5 ) cultures. By four days, however, some of the ICMs in
experimental cultures are significantly smaller than in the controls. As shown in
Table 1, by seven days (e.g.d. 11-5) 30-50% of the ICMs from experimental
blastocysts can be classified by phase-contrast microscopy as definitely abnormal
in their morphology according to the following criteria: small size, either the
absence of a central cavity or the presence of a small cavity containing dense
material, and multiple outer layers of endoderm cells, which often slough off
in clumps into the medium (Fig. 3 a, b). Some of these presumed *w5/*w5 ICMs
attached to the culture dish where the endoderm cells extended into flat sheets,
but these endoderm cells did not continue to grow beyond about 9 days in
culture.
In the electron microscope presumed + / + and + / / w 5 ICMs fixed on day 7
of culture (e.g.d. 11-5) have a single epithelial layer of endoderm cells separated
by large spaces containing mesodermal cells from the embryonic ectoderm,
which usually consists of a single layer of cuboidal or columnar cells surrounding
a large cavity (Fig. 3 c). In the case of presumed J w5 /r v5 ICMs fixed on day 7 of
culture, both light and electron microscope sections reveal that the outer
endoderm cells are producing large amounts of extracellular material which
accumulates between the endoderm and the epiblast (Fig. 3 b). The endoplasmic
reticulum of the endoderm cells is engorged with this secretory material. The
inner cells appear quite healthy and have many desmosomes and other types
of junctions, even when the cells show no obvious organization into an
epithelial layer.
DISCUSSION
In these experiments we have shown abnormal development of presumed
t°/t° inner cell masses cultured in vitro in the absence of trophectoderm (which
was removed during the immunosurgery procedure). By three days in culture
(e.g.d. 7-5) the presumed homozygous t° ICMs resemble the egg cylinders of
presumed t°/t° embryos of approximately six days of development in utero
(Gluecksohn-Schoenheimer, 1940). In both cases the presumed homozygous
mutants are much smaller than normal and have an irregular outer layer of
endoderm cells surrounding a disorganized mass of epiblast cells which do not
form a proamniotic cavity. In other experiments, Wudl & Sherman (Wudl et al.
1977; Wudl & Sherman, 1978) have reported that trophectoderm cells of
presumed t6/t6 blastocysts fail to endoreduplicate their DNA in culture to the
Cultured ICMs from t mutant mouse embryos
same extent as controls, although other properties of the trophectoderm cells
(such as the synthesis of specific enzymes) are apparently unaffected. Reduced
endoreduplication of DNA was also found in cultures of presumed homozygous
mutant trophectoderm vesicles, which had developed from dissociated blastomeres in the absence of an inner cell mass. Whatever the common factor
responsible for the defect in trophectoderm and ICM cells of cultured t°/t°
embryos, these results suggest that the t° mutation can be expressed independently in more than one cell type.
Inner cell masses from the cross +/fw6 x +/fwS develop in culture to a more
advanced stage than t° homozygotes. Subsequently, however, some of them
apparently fail to form either a fully expanded proamniotic cavity or mesoderm
cells, and show relatively luxuriant growth of endoderm cells and the accumulation of extracellular material between the endoderm and ectoderm (Fig. 3b),
all features consistent with the morphology of presumed homozygous mutants
in utero (Bennett & Dunn, 1958; Babiarz, personal communication). ICMs
with the abnormal morphology we have associated with the fw5/fw5 genotype
are not seen in cultures of ICMs from other late-acting t mutants {tw18 and fw73,
Hogan, unpublished observations). We therefore feel confident that the abnormalities we have imputed to presumed tw5/tM'5 ICMs are due to their genotype
and not to the culture conditions per se. However, the proportion of ICMs
scored as presumed homozygotes after 7 days in culture (average 37%) is
somewhat lower than the proportion of homozygotes expected in the experimental cross ( + /*w5 x + // w5 ) (approximately 50 %). This suggests that under
our conditions some homozygotes are developing at least to the same extent as
some of the normal ( + / + or + /fw6) ICMs. In this context it is interesting to
note that Bennett and Dunn (1958) described a class of homozygotes that
maintained sufficient viable embryonic ectoderm cells to permit some mesoderm
formation. At present we know nothing about the failure of presumed *w5/fw5
ICMs to develop normally in culture. It has been suggested that the Pv5 mutation
is expressed initially in embryonic ectoderm cells (Bennett & Dunn, 1958). Our
observations, as well as the finding that trophectoderm cells of fw5/*w5 blastocysts
behave the same in culture as control trophectoderm cells (Wudl & Sherman,
1978), are not inconsistent with this hypothesis.
This work was carried out in the Sloan-Kettering Cancer Center, and B.H. would like to
thank Dorothea Bennett for her generous support and encouragement, and for making the
facilities of her laboratory so freely available. All the members of the group, in particular
Karen Artzt, contributed to the work through many informative and enthusiastic discussions.
We would like to thank Cathy Calo and Steven Dunn for technical assistance.
Supported by NSF grant PCM 77-17835 and NIH CA-08748-13.
428
B. HOGAN, M. SPIEGELMAN AND D. BENNETT
REFERENCES
K., BENNETT, D. & JACOB, F. (1974). Primitive teratocarcinoma cells express a
differentiation antigen specified by a gene at the T-locus of the mouse. Proc. natn. Acad.
Sci., U.S.A. 71, 811-814.
BENNETT, D. (1975). The T-locus of the mouse. Cell 6, 441-454.
BENNETT, D., ARTZT, K., MAGNUSON, T. & SPIEGELMAN, M. (1977). Developmental interactions studied with experimental teratomas derived from mutants at the T/t locus in the
mouse. In Cell Interactions in Differentiation (ed. M. Kurkinen-Jaaskelainen, L. Saxen &
L. Weiss), pp. 389-393. Academic Press.
BENNETT, D. & DUNN, L. C. (1958). Effects on embryonic development of a group of
genetically similar lethal alleles derived from different populations of wild house mice.
/. Morph. 103, 135-158.
EPHRUSSI, B. (1935). The behaviour in vitro of tissues from lethal embryos. J. exp. Zool. 60,
197-204.
6 6
ERJCKSON, R. P. & PEDERSEN, R. A. (1975). In vitro development of f // embryos. / . exp.
Zool. 193, 377-384.
GLUECKSOHN-SCHOENHEIMER, S. (1940). The effect of an early lethal (/°) in the house mouse.
Genetics 25, 391-400.
HOGAN, B. & TILLY, R. (1978a). In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts. I. / . Embryol. exp. Morph. 45, 93-105.
HOGAN, B. & TILLY, R. (19786). In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts. II. / . Embryol. exp. Morph. 45, 107-121.
KEMLER, R., BABINET, C , CONDAMINE, H., GACHELIN, G., GUENET, J.-L. & JACOB, F. (1976).
Embryonal carcinoma antigen and the T/t locus of the mouse. Proc. natn. Acad. Sci.,
U.S.A. 73, 4080-4084.
6 6
NADIJCKA, M. & HILLMAN, N. (1975). Studies of t /t mouse embryos. / . Embryol. exp.
Morph. 33, 697-713.
SHERMAN, M. I. & WUDL, L. R. (1977). T-complex mutations and their effects. In Concepts
of Mammalian Embryogenesis. (ed. M. I. Sherman), pp. 136-234. Cambridge, Mass.: MIT.
SOLTER, D. & KNOWLES, B. B. (1975). Immunosurgery of mouse blastocysts. Proc. natn.
Acad. Sci. U.S.A. 72, 5099-5102.
WILEY, L. M., SPINDLE, A. I. & PEDERSON, R. A. (1978). Morphology of isolated mouse
inner cell masses developing in vitro. Devi Biol. 63, 1-10.
WUDL, L. R. & SHERMAN, M. I. (1976). In vitro studies of mouse embryos bearing mutations
at the T-locus: t^/tx\ Cell 9, 523-531.
WUDL, L. R. & SHERMAN, M. I. (1978). In vitro studies of mouse embryos bearing mutations
at the ^-complex: t6. J. Embryol. exp. Morph. 48, 127-151.
WUDL, L. R., SHERMAN, M. I. & HILLMAN, N. (1977). Nature of lethality of t mutations in
embryos. Nature, Lond. 270, 137-140.
ARTZT,
(Received 9 April 1980, revised 30 May 1980)