J. Embryo/, exp. Morph. Vol. 49, pp. 1-12, 1979
Printed in Great Britain © Company of Biologists Limited 1979
Developmental studies of the lethal gene Bid
in the mouse
I. Post-implantation development of the lethal homozygote
By G. LAWRENCE VANKIN1 AND ERNST W. CASPAR!2
From the Shanklin Laboratory of Biology, Wesley an University,
Middletown, Connecticut
SUMMARY
In matings of Bid/ + x Bid/+ mice a characteristic type of abnormal embryo is found on
days 6 and 7 after impregnation which dies at 8 days and accounts for about 25 % of all
living embryos. These embryos are regarded as the lethal Bid/Bid homozygotes. Before 6
days the embryos appear slightly retarded. Entodermal cells invade the yolk sac and the
trophectoderm does not fuse to the uterine decidua. Late on day 6 the yolk sac isfilledwith a
cap of unorganized cells of entodermal origin, surrounded by both a thick membrane of
non-living material, corresponding to Reichert's membrane, and external to this a continuous layer of trophectoderm; there is still almost no contact with uterine tissue. At 7 days
10 h amniotic folds are formed. Mesoderm appears occasionally but is not always present;
when it appears it does not grow out from its place of origin. Entodermal cells, particularly
in the proximal part of the extra-embryonic region, become polyploid. At the same time, the
trophectoderm makes contact with the uterine decidua and gives rise to primary giant cells.
Twelve hours later, the embryonic cells begin to degenerate, first in the region of the amniotic
fold and the mesoderm. The embryo dies shortly afterwards.
It is suggested that the primary target tissues affected by the Bid/Bid constitution are
trophectoderm and entoderm. Many but not all of the abnormal features appearing later
can be ascribed to insufficient nutrition of the embryo, due to failure of attachment of the
trophectoderm and thefillingof the yolk sac with entodermal cells.
INTRODUCTION
For a long time lethal genes have been a favourite object of study by developmental geneticists. Much information about early gene action in development
has been derived from the study of lethals. The older literature on this subject
has been repeatedly summarized, e.g. by Hadorn (1961) and Wright (1970) for
Drosophila, by Gluecksohn-Waelsch (1963 and earlier) and McLaren (1976) for
the mouse, and by Briggs (1973) for the axolotl.
The mutation Blind {Bid) arose spontaneously in the mouse strain BALB/Ci
1
Author's address: Department of Biology, Williams College, Williamstown, Massachusetts 01267, U.S.A.
2
Author's address: Department of Biology, University of Rochester, Rochester, New
York 14627, U.S.A.
2
G. L. VANKIN AND E. W. CASPARI
in 1949. It is expressed as open eyes at birth which develop into defective eyes
in the adult. The gene is dominant and lethal in homozygous condition, as has
been concluded from inability to obtain true breeding lines by inbreeding, from
the reduction in litter size in Blind x Blind crosses, and from the segregation
ratios obtained from Blind x Blind crosses compared to Blind x normal matings.
The development and penetrance of Bid in heterozygotes has been described by
Watson (1968). The mutation has been located on chromosome 15, 14-5 crossover units to the left of bt (Teicher & Caspari, 1978).
The present paper reports the development of the Bid homozygote as studied
with morphological methods. It is intended to show by direct observation that
the homozygote is lethal, a fact concluded previously from indirect evidence,
and to describe the developmental defects leading to death. Furthermore, this
morphological investigation is intended to serve as a basis for the study with
more analytical methods, such as use of the electron microscope, culture of
early embryos and chimera formation. A preliminary abstract of part of these
observations has appeared earlier (Vankin, 1956).
MATERIALS AND METHODS
As already mentioned, Bid arose in strain BALB/Ci which had been inbred
since at least 1938. Bid was kept mainly by outcrosses to BALB/Ci, though in a
few early generations Blind progeny from crosses Blind x Blind were used. The
frequent outcrosses to BALB make it certain that the two strains remained
isogenic except for the mutant gene Bid.
In order to obtain timed lethal embryos Blind animals were mated inter se
and the females examined for mating plugs at 8 h intervals. The postconception
(p.c.) age of the embryos was thus determined to within ±4 h.
Pregnant females were killed by cervical dislocation at different times after
copulation, and their uteri immediately dissected out in warm isotonic saline.
Decidual capsules or embryos removed from them were fixed in Bouin's,
embedded in Fisher Tissuemat, sectioned at 8 /im and stained with hematoxylin
and eosin. Some embryos were fixed in Zenker's, cleared in dioxan and stained
with congo red as recommended by Fekete, Bartholomew & Snell (1940). The
use of congo red as counter-stain aids in the distinction between embryonic and
extra-embryonic ectoderm in younger embryos.
Wherever possible, all embryos of each litter were serially sectioned and
mounted on slides. Both sagittal and frontal sections of the egg-cylinder stage
and later embryos were made. In this way, normal and lethal embryos of the
same age and the same litter could be compared. Adox KB-14 film was used for
the photomicrographs.
Lethal gene Bid in the mouse
Table 1. Results of dissections offemales for mat ings Blind x Blind
Total
Age of litters
in days p.c.
6^-7
7-8
8-8i
9-9i
104-19
Total
Embryos
litters
of embryos
Normal
Abnormal
Resorbed
6
11
4
4
5
30
51
90
35
36
48
260
37
63
27
22
30
179
11
23
2
0
0
36
3
4
6
14
18
45
RESULTS
1. The time of death of the homozygote
Since at the outset of the study the time of death of the lethal homozygotes
was unknown, fetal litters from Blind*. Blind crosses were grossly examined for
normal, abnormal, and dead and resorbed embryos starting with a litter shortly
before birth, 19 days p.c. From this litter we worked backwards in time to
progressively younger stages until consistent evidence for living abnormal
embryos was found. Once these abnormal embryos were identified they could be
subjected to closer histological study.
The results of the survey are shown in Table 1. The Table shows that of 141
embryos dissected before 8 days p.c. 100 were normal, 34 alive but abnormal,
and 7 were dead and resorbed. It will be shown in the next section that the
abnormal embryos show a characteristic and consistent pattern of abnormalities.
At gross observation, they are distinguished from their normal litter-mates by a
strong reduction in size. The abnormal embryos make up 25-4% of the total
number of living embryos, a proportion agreeing well with the expectation for
the frequency of homozygotes. The nature of the early resorbed embryos cannot
be determined. Adding them to the abnormal embryos raises the proportion of
abnormals to 29 %, suggesting that some of the early resorbed embryos may
have died for reasons other than homozygosity for Bid.
In the litters 9-19 days p.c. only normal and resorbed embryos were found
In this context, 'normal' includes embryos with incomplete eyelids which were
found in the 19-day-old litter and which represent the Bld/+ heterozygotes
(Shapiro, 1955; Watson, 1968). The percentage of resorbed embryos in all older
litters is 38-1. Most of the embryos dying after 9 days of gestation probably are
not Bid homozygotes since the expected proportion die on the ninth day, and
the characteristic abnormal embryos are not found later on. Many authors have
reported sporadic deaths in the development of genetically normal rodent
embryos. And Bld/+ embryos are expected to be among those dying after
9days/?.c. since Shapiro (1955) found that embryos from Bid/ + x + / + matings
show a slightly higher mortality than those from + / + inter se matings.
4
G. L. VANKIN AND E. W. CASPARI
In litters of 8-8^ days p.c, 27 normal, and a total of 8 (or 22-9 %) abnormal
and resorbed embryos were found. Six of these embryos were resorbed and the
other two were not; but it could not be ascertained whether they were still alive
since they were damaged and not preserved for histological study.
The data show that at about 8 days p.c. the characteristic abnormal embryos
are dying or dead. They are assumed to represent the Bid/Bid homozygotes
because they occur only in litters Blind x Blind and in these litters account for
about 25 % of the embryos.
2. Morphology of lethal embryos
The normal litter-mates of abnormal embryos have been checked against
descriptions in the literature to verify their age. Our 5 day 20 h normal embryos
represent a somewhat later stage than the 5 day 12 h embryo pictured in Snell
& Stevens (1966, fig. 12:8) and are at about the same stage as Theiler's (1972)
stage 9 (6^- days). Our 6 day 20 h stage corresponds to SneJl & Stevens' 6 day
13 h embryo (fig. 12:10) and to Theiler's stage 10 (7 days). Our 7 day 10 h stage
is similar to Snell & Stevens' 7 day 6 h embryo (fig. 12:13) and to Theiler's
stage 11 (7-^ days). In general the ages of our normal embryos correspond more
closely to Snell & Stevens' (1966) stages.
At about 6 days of gestation there already exists a difference between normal
and abnormal embryos. The abnormal embryos of Figs. 2 and 3 are shorter and
smaller than their normal litter-mates (Fig. 1) and resemble superficially fig.
12:7 of Snell & Stevens (1966), an embryo from the sixth day. In the abnormal
embryos, embryonic ectoderm surrounds the proamniotic cavity and is clearly
distinct from the extra-embryonic ectoderm. Entoderm surrounds the yolk
cavity in a normal manner, but the distal entoderm cells are round rather than
flattened. Furthermore, cells which may be derived from both the distal and the
proximal entoderm are seen within the yolk cavity. The trophectoderm cells
are distinct from the distal entoderm but do not invade the uterine decidua.
Instead, the parietal wall of the yolk cavity, consisting of trophectoderm and
distal entoderm, is surrounded by a cavity filled with cell debris. This external
cavity has apparently arisen by digestion of uterine tissue and, because of its
presence, the trophectoderm is not in normal contact with maternal tissue. In
the embryo of Fig. 3 the ectoplacental cone also is not in contact with uterine
tissue. Distal entoderm and trophectoderm are separated along part of their
facing surfaces, and the trophectoderm is attached to uterine tissue in some
places, though not as extensively as in normal embryos of the same age.
Fig. 5 shows a parasagittal section of an abnormal embryo 6 days 20 h old.
It is typical of the abnormals found at this stage, being much smaller and less
developed than its normal Jitter-mate of Fig. 4. The normal embryo at this time
shows the formation of mesoderm and the outgrowth of the amniotic fold
which separates the amniotic cavity from the ectoplacental cavity. The proximal
entoderm in the normal embryo is columnar in the extra-embryonic region and
Lethal gene Bid in the mouse
Fig. 1. Normal embryo, 5 days 20 hp.c. e = entoderm, y = yolk sac cavity, x 230.
Fig. 2. Bid/Bid embryo, 5 days 20 h p.c, same litter as Fig. 1. e = entoderm,
y = yolk sac cavity, c = cavity surrounding parietal yolk sac, t = trophectoderm.
x230.
Fig. 3. Bid/Bid embryo, 5 days 20 hp.c, different litter than Figs. 1 and 2. x 230.
Fig. 4. Noimal embryo, 6 days 20 h p.c. f = amniotic fold, m = mesoderm, e =
entoderm, x230.
G. L. VANKIN AND E. W. CASPARI
Fig. 5. Bid/Bid embryo, 6 days 20 h p.c. e = entoderm, / = trophectoderm, c =
cavity surrounding parietal layer of yolk sac. x 230.
Fig. 6. Embryonic part of Fig. 5. x 460.
Fig. 7. Bid/Bid embryo, 7 days 10 hp.c. e = entoderm, eg = entodermal giant cell,
/ = amniotic fold, g = primary giant cells, x 230.
Fig. 8. Bid/Bid embryo, 7 days 16 h p.c. e = entoderm, m = mesoderm, / =
amniotic fold, x 230.
Lethal gene Bid in the mouse
7
undergoes a gradual transition to cuboidal and finally to squamosal epithelium
in the embryonic portion. The abnormal embryo at the same age (Fig. 5) is
smaller than normal, though it has clearly grown compared to the earlier stage
(Figs. 2 and 3). The embryo of Fig. 5 appears compressed and slightly bent,
possibly a fixation artifact, and is considerably retarded compared to its normal
litter-mates. The ectoderm is clearly divided into embryonic and extraembryonic regions. There is no indication of mesoderm or amniotic fold
formation in abnormal embryos of this age. The entoderm cells appear slightly
vesicular but alive; they do not show the normal differentiation into columnar,
cuboidal and squamosal epithelium. Below the egg-cylinder's antimesometrial
pole there appears a large irregular accumulation of cells. It corresponds by
position to an extension of the yolk cavity that has been filled with cells, some of
which are presumably derived from the proximal entoderm. This cell mass is
variable in size and is frequently smaller than it is in Fig. 5; but it is always
present at this stage. The numerous cells of this cell mass are embedded in a
characteristic non-cellular, eosine-staining substance. The eosinophilic condition of this material is identical with the usual staining of Reichert's membrane
in histological preparations of normal embryos. The cell mass is surrounded
exteriorly by a thick structureless membrane which according to its position and
staining properties is assumed to represent Reichert's membrane. On the outside
of this membrane large rounded cells are seen which, according to their position
(compared to the younger embryos of Figs. 2 and 3), presumably represent
trophectoderm. The whole structure is surrounded by a cavity which is filled
with digested uterine material. Higher magnification of the same embryo
(Fig. 6) shows that the ectoderm cells look healthy and show mitoses. The
proximal entoderm cells in the extra-embryonic region are rounded and not
columnar and the distal entoderm cells are not squamosal. In the embryonic
region, the proximal and distal entoderm cells are continuous with the unorganized cell mass.
By 7 days 10 h p.c. normal embryos have grown considerably. Amnion and
chorion, and the resulting cavities, amniotic cavity, extra-embryonic coelom and
ectoplacental cavity, are clearly established. The mesoderm shows extensive
differentiation, the primitive streak being conspicuous, the allantois growing
into the" exocoelom and the head process becoming apparent. All of these
structures are absent in the abnormal embryos at the same age. They are now
much smaller than their normal litter-mates, even though they have definitely
grown compared to abnormal embryos at 6 days 20 h. An abnormal embryo at
7 days 10 h p.c. is shown in Fig. 7.
One feature distinguishing the embryo in Fig. 7 from earlier abnormal
embryos is that at the antimesometrial pole the parietal wall of the yolk cavity
has become fused with the maternal decidua while laterally this contact has not
been accomplished. Primary giant cells can be clearly seen in the maternal tissue
and possibly also in the debris surrounding the egg cylinder laterally. This
8
G. L. VANKIN AND E. W. CASPARI
establishment of contact with the uterine decidua is frequently found in abnormal embryos at late stages. The cell mass at the yolk cavity's antimesometrial end is still present but smaller than in Fig. 5. This may or may not be
connected with the contact established with maternal tissue. As mentioned
earlier, the size of the cell mass is rather variable at 6 days 20 h, and similar
variability is found in embryos 7 days 10 h old and older. Thus, embryos with
cell masses larger than in Fig. 7 are found up to the second half of the eighth
day.
The embryo of Fig. 7 shows well developed amniotic folds, dividing the
proamniotic cavity into an amniotic cavity and an extra-embryonic portion.
The ectoderm looks healthy and shows numerous mitoses. It is, in the region of
the posterior amniotic fold, separated from the entoderm but no intermediate
layer corresponding to mesoderm can be distinguished in the majority of the
lethal embryos. But there occur individuals in which a wedge of cells separate
from ectoderm and entoderm develops in the region corresponding to the
primitive streak (see Fig. 8). This presumed mesoderm layer of lethal embryos
remains confined to a limited region, roughly the midline at the dorso-posterior
extremity of the embryo. It does not migrate around the circumference of the
embryo as the mesoderm does in normal embryos.
The syndrome of abnormalities exhibited at 7 days 10 h in lethal embryos
includes additional gross disturbances. The cells of the proximal entoderm,
particularly those adjacent to the extra-embryonic ectoderm, are conspicuously
enlarged and somewhat disorganized. These giant cells are not present in
earlier lethal embryos, as can be seen in Figs. 5 and 6, but are a consistent
feature of lethal embryos from 7 days 6 hp.c. on. The argument for regarding
these giant cells as entodermal in origin is based on their position relative to the
ectoderm. It appears as if both proximal and distal entoderm are involved.
These giant cells are polyploid, as suggested by a cell in the region of the
amniotic fold which was found in mitosis. This cell displayed a tetrapolar
spindle and a large number of indistinct possibly shattered chromosomes.
Barlow & Sherman (1972, fig. 4) have shown that in giant trophoblast cells
there is a good correlation between nuclear size and DNA content. Therefore
we compared the volumes of proximal entodermal cells in normal and lethal
embryos. Determinations of nuclear volumes were performed by making
camera lucida drawings of each nucleus and measuring the long and short
diameter with a millimeter ruler. The measurements in millimeters were converted into microns, and the volume of the nucleus calculated. Only nonsquamosal proximal entoderm cells were used for measurement in both normals
and lethals. Fifty cells were measured in a group of lethal embryos between the
ages of 7 days 6 h and 7 days 20 h p.c, and 50 cells from normal litter-mates
were used as controls.
The results are presented in the form of histograms in Fig. 9. The nuclei from
normal cells show a fairly normal distribution of volumes with a mean of
Lethal gene Bid in the mouse
30
20
y. 10
200
pin3
400
200
400
600
800
1000
1200
Aim3
Fig. 9. Volumes of nuclei of entodermal cells from normal (A) and Bid/'Bid (B)
embryos 7 days 6 h to 7 days 20 h p.c.
188 ± 9-8 /«n3. The nuclei of the lethal cells show considerable variability, and
there is some overlapping between the largest normal cells and the smallest
lethal cells. But the majority of the lethal cells are outside the range of the
normals and their mean is 603 ± 41-6 /tm3. A similar study was carried out on
entoderm cells of normal and lethal embryos less than 7 days old. In this
material of 25 lethal and 25 normal nuclei no significant differences between the
mean volumes of the two samples were found. In both normals and lethals the
average nuclear volume of entoderm cells is about 110/*m3 and both samples
are fairly normally distributed about the mean. There are, however, in lethal
embryos of this age, a very few individual cells which have an enlarged nucleus
compared to the normals of the same age. These cells show a maximum nuclear
volume of 300 /tm3 and are thus still smaller than the enlarged nuclei on the
eighth day. It is not clear whether these giant cells arise by endomitosis or by
cell fusion. But they start appearing late on the seventh day and are fully
developed by 7 days 6 h.
Lethal embryos shortly before the time of death show no further advance
beyond the development at 7 days 10 h, as can be seen by comparing Fig. 7 with
Fig. 8. The amniotic fold in Fig. 8 has not undergone the expansion and
thinning characteristic of normal embryos. Instead, the first signs of degeneration and death of lethal embryonic tissue become evident in the amniotic fold.
This is already indicated by the appearance of metachromatic granules in the
cells of the amniotic fold in Fig. 7. In Fig. 8, most cells of the amniotic fold
appear degenerate, and the degeneration starts to expand into the embryonic
ectoderm. Degeneration is also seen in the cells of the lateral amniotic constrictions at this stage as can be seen in frontal sections.
Fig. 8 shows an embryo which has developed a small wedge-shaped group of
cells between ectoderm and entoderm at the level of the amniotic fold. This cell
10
G. L. VANKIN AND E. W. CASPARI
layer, previously identified as mesoderm, does not develop further but shows
signs of degeneration at 7 days 16 h. The ectoplacental cone and the cell mass at
the yolk cavity's antimesometrial end still look healthy, and the embryo now
appears well attached to the uterine tissue. The primary giant cells are quite
conspicuous.
The series of events described in Figs. 1-8 may be regarded as typical of the
lethal Bid/Bid embryos. One small embryo did not yet show any indication of
cellular degeneration at 7 days 22 h. This embryo had a fairly normal entoderm,
no giant cells and no disorganized cell mass. A large fold of ectoderm and
entoderm, which may represent the head fold, had formed. No indication of
mesoderm was found. Even though this embryo had survived, without degeneration, beyond the usual time of death of abnormal embryos, it is highly
abnormal and would probably have died at a somewhat later stage than is usual
for Bid homozygotes. This embryo is possibly a Bid/Bid embryo which has
survived the usual time of death, a 'breakthrough' or 'escaper' (Landauer,
1932; Hadorn, 1961).
DISCUSSION
The i?/d homozygote shows a complex pleiotropic pattern of defects affecting
different structures and germ layers. Similar defects have been found in other
early lethal mutants of mice, but in different combinations and with different
consequences.
Of the abnormalities in Bid observed before day 6 p.c. retarded growth is
almost universal among early embryonic lethals. Failure of attachment of the
trophoblast to the decidua has been described for Av/Av by Eaton & Green
(1973) and for tw™/tw™ by Spiegelman, Artzt & Bennett (1976). The former
homozygote dies shortly after implantation, but tw™ resembles Bid in several
respects: the attachment of the parietal layer of the yolk sac to the decidua is
delayed and incomplete, and primary giant cells do not appear in the decidua
or appear late. In both mutants, the egg cylinder is surrounded by a cavity
filled with debris, probably indicating excessive destruction of uterine tissue.
A second early abnormality in Bid/Bid embryos is the disorganization of the
entoderm. This is a frequent feature in early embryonic lethals. In
cm/cm
embryos, Lewis, Turchin & Gluecksohn-Waelsch (1976) found that cells of the
parietal entoderm become disorganized and migrate into the decidua forming
a dense aggregate of cells similar to the aggregate at the yolk cavity's antimesometrial end in Bid/Bid. In Bid and cm homozygotes a substance similar to
Reichert's membrane is deposited between the cells forming the dense aggregate.
Since Reichert's membrane is assumed to be derived from entoderm (Snell &
Stevens, 1966), this observation supports the identification of the disorganized
cell accumulation in cm and Bid as entodermal.
There are no obvious abnormalities in the Bid/Bid ectoderm. Bid/Bid
embryos form amniotic folds which may fuse centrally. They do not form a
Lethal gene Bid in the mouse
11
complete primitive streak and mesoderm, though some abnormal embryos form
an abortive mesoderm in the region of the posterior amniotic fold. The facts
that amniotic folds appear regularly but mesoderm only occasionally indicate
that the lack of mesoderm in Bid is a secondary consequence of other processes,
probably the general inhibition of growth.
The sudden appearance of polyploid giant cells in the extra-embryonic
entoderm is specific for Bid and has not been found in any other mutant. The
relationship of the entodermal giant cells in Bid to the other pleiotropic effects
of this gene is not clear.
Spiegelman et al. (1976) have suggested that the primary effect of twlz is an
interference with the interaction between trophectoderm and maternal decidua.
A similar interpretation may be suggested for Bid which in its effects on trophectoderm differentiation and primary giant cell migration resembles tw™.
But this assumption leaves unexplained the migration of entoderm cells into the
yolk cavity, a process not found to the same extent in tw™. Lewis et al. (1976)
refuse to speculate on the primary effect of cm since more than one primary
effect may be assumed. This possibility is suggested by the fact that cm is a
deficiency which may include loci adjoining c. This cautious attitude may be
extended to other lethals including Bid. While we have to assume that each gene
has a unitary primary function which is changed in a mutant in a specific way,
it seems impossible to explain the complex pleiotropic patterns frequently found
in mutants from morphological observations alone.
We thank Dr Uzi Nur, Dr Rudolf E. Haffner and Miss Ingbritt Blomstrand for valuable
aid and advice in the photography.
This investigation was supported in part by a grant to Williams College from the Whitehall
Foundation.
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{Received 8 May 1978, revised 1 September 1978)