/. Embryol. exp.'Morph. Vol. 48, pp. 127-151, 1978
Printed in Great Britain © Company of Biologists Limited 1978
127
In vitro studies of mouse embryos
bearing mutations in the T complex: t6
By LINDA R. WUDL 1 AND MICHAEL I. SHERMAN 2
From the Roche Institute of Molecular Biology, New Jersey
SUMMARY
Cultured blastocysts homozygous for the /6 mutation lose their inner cell mass within a
few days of attachment to the culture dish. At about the same time it becomes apparent that
putative ^-mutant trophoblast cells and their nuclei fail to enlarge to the degree of their
normal counterparts. These abnormalities in mutant embryos are reflected by an abrupt
drop on the seventh equivalent gestation day in the rate of increase of /?-glucuronidase
activity. The failure of t6/t6 trophoblast nuclei to enlarge normally appears to be due partially
to endoreduplication at a slower rate than normal trophoblast nuclei and partially to premature cessation of DNA synthesis. Analyses indicate that this abnormality is not reversed
when mutant embryos are placed in chimeric association with normal ones. Trophoblast
outgrowths from mutant and normal trophectodermal vesicles are similarly distinguishable
by differences in outgrowth and nuclear size as well as DNA content and synthesis. Despite
the fact that tG/t6 embryos and trophectodermal vesicles are phenotypically different from
normals from early times in culture, the trophoblast cells in the mutant structures acquire
and continue to produce two enzymes characteristic of trophoblast differentiation, A5,3/?hydroxysteroid dehydrogenase and plasminogen activator.
On the basis of these and previous observations, we propose that the primary effect of the
/6 mutation is to cause a metabolic lesion which kills inner cell mass cells relatively quickly
but which has a more gradual effect upon trophoblast cells. The fact that phenotypically
recognizable t^/t* trophoblast cells can survive for several days before dying makes this a
potentially useful system in which to search for the t6 gene product(s).
INTRODUCTION
Recessive lethal t mutations in the mouse fall into six 'complementation'
groups which are characterized by the time and stage of embryonic development
at which lethality is observed (see Sherman & Wudl, 1977). Previously we have
presented evidence for the 'generalized cell-lethal' nature of at least three of
the six types of lethal t mutations (Wudl & Sherman, 1976; Wudl, Sherman &
Hillman, 1977). That is, all cells in the embryo are adversely affected by the
mutation, even under conditions which minimize the need for embryonic
organizational integrity. We also observed that homozygous t12-, ?6- and tw5mutant embryos behave in culture as they do in vivo, with disruption of development occurring abruptly and at a time and stage determined by the individual
1
Author's address: Department of Biochemistry and Drug Metabolism, Hoffman-La
Roche, Inc., Nutley, New Jersey 07110, U.S.A.
2
Author's address for reprints: Roche Institute of Molecular Biology, Nutley, New Jersey,
07110, U.S.A.
9-2
128
L. R. WUDL AND M. I. SHERMAN
mutation. This, therefore, suggests that the lethal effects of t mutations are
linked rather closely to the 'developmental clock'. However, these observations
do not allow us to decide whether t mutations disrupt the developmental programme directly or indirectly by their effects upon some basic metabolic process.
Consequently, we are attempting to address this question by studies on early
acting t mutations (causing death between the 4th and 8th days of gestation)
aimed at closer scrutiny of the events occurring at or near the time at which
abnormalities first become apparent.
Homozygous t% embryos in vivo are grossly abnormal by the 7th day of
gestation (Nadijcka & Hillman, 1975; see also Gluecksohn-Schoenheimer,
1940, for morphological studies on the related mutation, t°). Recently, we
described the lethal effects of this mutation on embryos grown in an ectopic
site and in vitro (Wudl et al. 1977; Sherman & Wudl, 1977). We found that in
culture, putative ?6/f6 blastocysts hatch and subsequently attach to the substratum in a manner indistinguishable from control ( + / + or + /f6) blastocysts.
Thereafter, however, the behaviour of presumptive t®/t6 embryos departs from
that of their normal counterparts: soon after trophoblast cells begin to grow
out along the culture dish, necrotic cells are apparent in the mutant inner cell
masses (ICMs) and by the 9th EGD (equivalent gestation day, i.e. their age
had they remained in utero), these ICMs have completely disintegrated (Wudl
et al. 1977; see also Erickson & Pedersen, 1975). Furthermore, unlike trophoblast cells from + / + and + /t6 embryos, even exceptional ones that 'spontaneously' lose their ICMs, trophoblast cells in presumptive mutant embryos
fail to enlarge normally as the cultures develop; by the 1 lth EGD, some of the
mutant trophoblast cells have died, and many of the remaining cells appear to
be in the process of degeneration (Sherman & Wudl, 1977). That the presence
of ICM cells in control embryos and their absence in mutant embryos is not
responsible for the differential appearance of normal and mutant trophoblast
cells could be shown conclusively by constructing trophectodermal vesicles
(TVs), structures which lack ICM cells. Trophoblast cells in outgrowths from
6
t$/t6 j y s a r e distinguishable from those of + / + and + // TVs by the same
features observed for trophoblast cells outgrowing from blastocysts (Wudl et al.
1977).
In this report, we describe the results of more extensive studies of t& embryos
in vitro, investigations dealing with both the nature and time of expression of
this mutation. We have analysed the levels in mutant embryos of an enzyme,
/?-glucuronidase, that rises dramatically during early development (Wudl &
Chapman, 1976; Wudl & Sherman, 1976) in order to get some idea of when
cellular metabolism is first interrupted; we have also looked for the expression
by ?6-mutant embryos and TVs of plasminogen activator (Strickland, Reich &
Sherman, 1976; Sherman, Strickland & Reich, 1976) and A5,3/?-hydroxysteroid
dehydrogenase (3/?-HSD) (Chew & Sherman, 1975; Sherman & Atienza, 1977),
enzyme markers that are characteristic of trophoblast differentiation. Finally,
Studies on ^-mutant embryos
129
6 6
the small size of t /t trophoblast nuclei suggested to us that they were failing to
polyploidize normally (Sherman & Wudl, 1977; Wudl & Sherman, 1977); we
have therefore compared the DNA content of, and incorporation of thymidine
into DNA by, nuclei of phenotypically mutant v. normal trophoblast cells.
The ?6 mutation lends itself to such studies because its critical period coincides
with the time of marked differentiation of trophoblast cells and, while the
mutation affects these cells adversely, it is not immediately lethal to them. This
decreases the chance of observing alterations which are artifacts resulting from
cell death. A further advantage of the t6 system is that mutant trophoblast cells
which have never been in contact with other embryonic cell types can be
identified and obtained. Therefore, we can largely eliminate any adverse effects
of improper cell-cell interaction as being causitive of abnormalities that are
observed. In short, we are attempting to reduce the complexity of the 7-mutant
experimental system from one involving observations on dying embryos in vivo
to one dealing with a single living cell type in vitro.
METHODS AND MATERIALS
Materials
[3H]thymidine, [3H]uridine and [3H]progesterone were purchased from
New England Nuclear, Inc., Boston, Mass. NCTC-109 culture medium and
fetal calf serum were purchased from Microbiological Associates, Bethesda,
Md. Progesterone and pregnenolone were purchased from Steraloids, Inc.,
Pawling, N.Y., and recrystallized prior to use. Antiserum to progesterone (lot
S257, no. 2) was purchased from Dr Guy Abraham, Harbor General Hospital,
Torrance, Calif. 4-Methylumbelliferyl-/?-D-glucuronic acid was purchased from
Sigma Chemicals, St Louis, Mo. Basic fuchsin for the Feulgen reaction was
purchased from Allied Chemical, Morristown, N.J., and adsorbed with activated
charcoal to give a straw-yellow color prior to use. Fibrinogen, bovine, fraction I,
was obtained from Calbiochem, La Jolla, Calif., and was purified by (NH4.)2SO4
and ethanol precipitations as described previously (Beers, Strickland & Reich,
1975). Thrombin (topical) was purchased from Parke-Davis, Avon, Conn.
Pregnant mare serum (Gestyl) and human chorionic gonadotrophin (Pregnyl)
were purchased from Organon, South Orange, N.J. Coomassie brilliant blue
R-250 was purchased from Inolex Corp., Glenwood, 111. NTB-2 Nuclear Track
Emulsion was obtained from Eastman Kodak, Rochester, N.Y. G. T. Gurr
neutral mounting medium was purchased from BioMedical Specialties, Santa
Monica, Calif. Culture vessels were from Falcon, Oxnard, Calif., except for
8-chamber, fixed gasket tissue culture slides which were obtained from LabTek Products, Napierville, 111.
130
L. R. WUDL AND M. I. SHERMAN
Mice
T/t* tailless mice were obtained originally from Dr N. Hillman, Temple
University, Philadelphia, Pa. Males were mated with SWR/J (wild-type for
T-complex genes) females (Jackson Laboratory, Bar Harbor, Maine). The offspring were checked for tail size and from these observations, it was observed
that the male transmission frequency for the t6 allele was approximately 80 %
(Wudl et al. 1977). Normal-tailed offspring ( + /?6) were used in the experiments
to be described. In the experimental cross, + /tb mice were mated inter se; the
expected proportion of genotypes of theblastocysts obtained was + / + = 10 %;
+ /t« = 50 %; te/tG = 40 %. In the control cross, + / + (SWR/J) females were
mated with + /? 6 males. The expected proportion of genotypes was: + / + =
20 %; + / ' 6 = 80 %. Females were superovulated (Runner & Palm, 1953) prior
to mating. The day of observation of the sperm plug is considered thefirstday
of pregnancy.
Collection and culture of embryos
Late morulae and early blastocysts were collected on the fourth day of
pregnancy and cultured individually in wells of Falcon Microtest I dishes containing NCTC-109 medium supplemented with antibiotics and 10 % heatinactivated fetal calf serum as described by Wudl & Sherman (1976). For
Feulgen staining and autoradiography, embryos were cultured in groups of five
in each compartment of an 8-chamber Lab-Tek slide. After the incubation
period, the chambers were removed and the gaskets were scraped off with a
scalpel blade.
Trophectodermal vesicles (TVs) were prepared according to the procedure of
Tarkowski & Wroblewska (1967) with modifications described by Sherman
(1975 a). Generally, embryos were removed at the two-cell stage, cultured for
one day in preimplantation culture medium (Goldstein, Spindle & Pedersen,
1975) and then the blastomeres were disaggregated and cultured individually
(from 4-cell embryos) or in pairs (from 8-cell embryos) in the same medium.
After 2 days the resulting structures were inspected under an inverted phase
optics microscope and TVs were collected, transferred to supplemented NCTC109 medium and cultured as were blastocysts. Phenotyping of the resultant
trophoblast outgrowths has revealed that TVs from t6/t6 embryos survive
initially as well as do their + / + or +/7 6 counterparts: in three experiments
a total of 130 experimental cross TVs were analyzed and 41 % had a tG/tG phenotype, very close to the expected value of 40 %.
Chimeric embryos were made at the 8-cell stage between pairs of embryos
obtained from the experimental cross (Wudl & Sherman, 1976).
Embryonic development in culture is referred to in terms of equivalent
gestation day (EGD), i.e. the age of the embryos had they been left in utero.
EGD terminology is also used for TVs. It should be noted that TVs of a given
Studies on ^-mutant embryos
131
EGD are developmental!)? retarded by approximately 1 full day when compared
with blastocysts on the same EGD. This is because it takes about a day longer
overall in comparison with blastocysts for disaggregated blastomere growths to
pass through the initial developmental stages, i.e. cavitation, attachment to the
culture dish, and outgrowth. Normal and mutant blastocysts could be distinguished phenotypically on the 7th EGD, but TVs could only be phenotyped on
the 8th EGD.
Nuclear and outgrowth measurements
Blastocyst or TV outgrowths were photographed (165 x magnification) with
a Wild inverted phase-optics microscope equipped with a Polaroid camera
attachment. Average nuclear diameters were measured from the photographs
according to the formula dav = ^(dt + dg), where dt and ds are the largest and
smallest diameters, respectively. Outgrowth areas were measured on the photographs with a planimeter.
DNA analyses
Blastocysts or TVs were fixed and stained by the Feulgen reaction as described
by Barlow & Sherman (1972). Coverslips were affixed to the slides with neutral
mounting medium. DNA contents were determined by microfluorometry with
the aid of a Leitz MPV 1 microfluorometer. Excitation and emission wavelengths were 560 and 627 nm, respectively. Machine units were converted into
ploidy (C) values by measurements of liver nuclei, which contain 2, 4 and 8
times the haploid amount of DNA (Barlow & Sherman, 1972).
A utoradiography
Blastocyst or TV cultures were incubated in 0-5 ml volumes in chamber
slides in the presence of [3H]thymidine (24 h) or [3H]uridme (4 h) at 1 /^Ci/ml
and a final specific activity of 10 mCi/mmole. After the incubation period the
cultures were washed, fixed, treated with trichloroacetic acid and autoradiographed as described previously (Sherman & Atienza, 1975). Slides were
exposed for 7 days at 4 °C, developed with Kodak Dektol developer for 2 min,
and stained with Giemsa.
Enzyme analyses
/?-glucuronidase assays were carried out by measuring the release of 4-methylumbelliferone from 4-methylumbelliferyl-/?-D-glucuronic acid, as described by
Wudl & Sherman (1976). Plasminogen activator assays were done by the fibrinagar overlay procedure (Strickland et ah 1976), i.e. blastocysts or TVs cultured
in 35 mm culture dishes were covered with a mixture of agar, medium, fibrinogen,
thrombin and acid-treated fetal calf serum. The thrombin converts the fibrinogen
to a fibrin clot. Plasminogen activator, secreted by the cells, will locally convert
plasminogen in the serum to plasmin. The plasmin in turn breaks down the
132
L. R. WUDL AND M. I. SHERMAN
fibrin in the area. The result is a zone of lysis surrounding plasminogen activator
positive cells; when the undegraded fibrin is stained with Coomassie brilliant
blue, the lysis zones are detectable as clear halos around the cells. A5,3/?Hydroxysteroid dehydrogenase (3/?-HSD) activity was determined on single
blastocysts cultured in wells of a Microtest dish in NCTC-109 medium supplemented with serum absorbed with dextran-norit to remove endogenous steroids
(Salomon & Sherman, 1975). On the 9th EGD the cultures were washed and
each blastocyst was fed with 15 /d of this medium containing pregnenolone at
a concentration of 1 /^g/rnl. Twenty-four hcurs later 10 jn\ of culture medium
were collected and monitored for the presence of progesterone by radioimmunoassay (Marcal, Chew, Salomon & Sherman, 1975).
RESULTS
fl-Glucuronidase activity in ^-mutant and normal embryos
Embryos derived from control (+ / + x + // 6 ) and experimental ( + /t6x
+ /tG) crosses were cultured singly and individual enzyme activities were
measured from the 4th through the 9th EGD. The average enzyme activities
for embryos from both the experimental and control crosses were determined
for each day (Table 1). It is apparent that the overall average enzyme activities
in both crosses rise markedly throughout most of the assay period. Since, due
to transmission frequency distortion, approximately 40 % of embryos in the
experimental population are homozygous t6 mutants (Wudl et ah 1977), both
the experimental and control populations were divided into subpopulations
containing 60 % of the embryos with the highest enzyme activity and the
remaining 40 % with the lowest activity. So long as enzyme activities of the total
population represent a unimodal distribution about the mean activity, the ratio
of average activities of the two subpopulations should remain constant. That
this is the case for the embryos in the control cross is illustrated in Table 1 and
Fig. 1. On each of the days studied, the average of the individual /?-glucuronidase
activities in the 'high 60 %' subpopulation is approximately twice that found
for the 'low 4 0 % ' subpopulation (Table 1). Thus, the rates of increase in
enzyme activity are the same for both groups (closed and open circles, Fig. 1).
The situation for embryos in the experimental cross is initially the same as
that for the control cross, but only to the 6th EGD. Up to and including that
time, the enzyme activities in the high 60 % group are approximately twice those
of the low 40 % subpopulation (analyses of the distributions of individual
enzyme activities in the total control and experimental crosses indicate that
on the 6th EGD, the variances are not significantly different; P > 0-1). Thus,
4th to 6th EGD homozygous mutant embryos cannot be distinguished from
heterozygous and wild-type counterparts on. the basis of /?-glucuronidase activity.
After the 6th EGD, the high 60 %/low40 % ratio in the experimental population
increases continuously (Table 1). Fig. 1 illustrates that the high 60 % sub-
Studies on ^-mutant embryos
133
Table 1. fi-glucuronidase activities in normal and t^-mutant embryos
EGD
No. of
embryos
Total
High 60 %
Low 40 %
High 60 %
Low 40 %
Control cross ( + / + x +/tG)
Average enzyme activity (x 10- 13 mole/hour)
4
5
6
7
8
9
19
31
65
42
34
33
0-9
2-6
9-3
29-3
74-7
93-4
0-7
1-6
60
17-8
49-2
60-4
11
3-4
11-5
37-1
92-6
114-9
6
1-6
2-1
1-9
2-1
1-9
1-9
6
Experimental cross (4-A x+A )
Average enzyme activity (x 10~13 mole/hour)
4
5
6
7
8
9
26
40
74
53
40
33
11
6-8
12-5
28-0
76-2
88-6
1-4
8-5
16-4
38-3
115-7
135-9
0-7
4-2
6-9
12-4
16-8
15-8
20
2-0
2-4
31
6-9
8-6
Embryos from the appropriate cross were removed on the fourth day of pregnancy and
were cultured and analyzed individually for enzyme activity as described in Methods. The
resultant enzyme activities in each group were averaged as a total population. Also, the
highest 60 % of the values and the remaining values ('low 40 %') were individually averaged.
population continues to show increased enzyme activities with a rate paralleling
that of embryos in the control cross (the absolute enzyme activities in the
experimental cross are greater because these embryos were somewhat advanced
over those in the control cross at the beginning of the experiment). The rate
curve for the low 40 % population in the experimental cross, however, drops
off between the 6th and 7th EGD and remains subnormal through the 9th EGD.
Thus, the abrupt departure from normality of /?-glucuronidase levels in putative
?6-mutant embryos correlates with the time at which mutant ICM cells are
visibly degenerating and mutant trophoblast cells can first be distinguished
from normals by virtue of the smaller size of their nuclei.
Effects of the t6 mutation on trophoblast nuclear and outgrowth size and DNA
content
For these studies, we have analyzed trophoblast cells derived either from
blastocysts or from TVs. To demonstrate that all TVs from a single normal or
mutant embryo would behave similarly when cultured further, and that we
could dependably distinguish between mutant and normal outgrowths, we
followed the development of TVs derived from the same embryo. Although
disaggregated blastomeres can develop as miniblastocysts and non-integrated
134
L. R. WUDL AND M. I. SHERMAN
EGD
Fig. 1. /?-glucuronidase activities in cultured blastocysts from control and experimental crosses. The values are taken from the data in Table 1. # , Control cross,
high 60%; O, control cross, low 40%; A, experimental cross, high 60%; A,
experimental cross, low 40 %.
forms as well as TVs (Tarkowski & Wroblewska, 1967; Sherman, 1975 a), we
observed a number of cases in which all four pairs of 8-cellblastomeres developed
into TVs. Nuclear diameters were measured in 11th EGD outgrowths from two
such sets, one wild-type, the other phenotypically mutant. The data in Fig. 2
demonstrate first that the nuclear diameters in the wild-type outgrowths are,
on average, substantially larger than those in the presumptive mutants (30-2 v.
19-8 /tin); second, the range of nuclear diameters from one TV to another
within the same set is very similar. Thus, each TV behaves in a manner consistent
with the genotype of the original embryo.
We have also measured total culture areas of individual TVs from control
and experimental crosses on consecutive days from the 7th to 11th EGD. We
found differences between the two populations of TVs beginning on the 8th EGD
(Fig. 3, open v. filled circles). If we once again group the data from the experimental population into high 60 % and low 40 % subpopulations it is apparent
that beyond the 7th EGD, the average culture areas for the high 60 % resembles
Studies on tQ-mutant embryos
•
5
5
5
135
-
-
12
18 24
30 36 42
Nuclear diameter (/im)
48
Fig. 2. Diameters of trophoblast nuclei from pbenotypically t"-mutant and normal
trophectodermal vesicles. The different symbols in A represent values from each of
four phenotypically mutant TVs on EGD 11 derived from a single embryo. Those
in B are from TVs from a control cross embryo of the same age.
the total control population, whereas the low 40 % population undergoes only
a slight increase in average culture area over the period of study. This difference
is not due to continuous cell death in presumptive mutant TVs: comparisons
between nuclear numbers in control and experimental cross outgrowths show no
substantial differences over the 5-day period of analysis (Table 2).
In order to determine whether the differences in nuclear size of trophoblast
cells in normal v. mutant embryos reflect differences in their DNA content, we
have measured DNA levels in trophoblast nuclei from blastocysts and TVs by
means of a quantitative Feulgen stain coupled with microfluorometry. In an
initial series of experiments, we cultured blastocysts from a (+ /t6 x + // 6 ) cross
and measured DNA contents in trophoblast nuclei on the 11th EGD. Mutant
blastocysts were recognized by their absence of ICM cells and by their small
trophoblast nuclear and culture areas. For comparative purposes, we selected
phenotypically wild-type blastocysts that had relatively small ICMs (but normal
trophoblast nuclear and culture areas) in an attempt to minimize any effects of
ICM cells. The results are recorded in Fig. 4 and Table 3. We found that all
blastocysts contained trophoblast cells with polyploid nuclei. However, the
distribution of DNA contents in trophoblast nuclei of phenotypically mutant
and normal blastocysts is visibly different: in general, normal trophoblast nuclei
136
L. R. WUDL AND M. I. SHERMAN
35
_ l
l
i
i
i
i
i
i
i
_
A
30 -
-
25 -
_
V
012 HI20 -
15
-
p.
10 -
-
5
1 1
20
i
i
M i l l
1 1 1 1
B
15
i
10
_V
Y
L
5-
8
9
10
Hquivalent gestation day
Fig. 3
Jl
nu
l
i
16
-1
i i i it
64 .256
C
Fig. 4
Fig. 3. Outgrowth area measurements of trophectodermal vesicles derived from
control and experimental cross embryos. After individually cultured TVs had begun
to grow out along the culture dish, they were photographed for five successive days.
The area of each outgrowth was measured with a planimeter and corrected for the
magnification factor of the photograph. O, Average area of 16-17 TV outgrowths from
the control cross. • , Average area of 40-45 TV outgrowths from the experimental
cross. The experimental cross TV outgrowth areas have also been averaged for the
high 60 % (A) and low 40 % (A) values beyond the 7th EGD. Separation into the
two classes was not carried out on the 7th EGD because TVs are only beginning to
grow out at this stage and no difference is observed in the average outgrowth area of
experimental and control cross TVs.
Fig. 4. DNA contents of trophoblast cells from phenotypically normal and /"-mutant
blastocysts. Blastocysts from the experimental cross were cultured to the 1 lth EGD.
They were then fixed and stained as described in Methods. Embryos were phenotyped
using a low power microscope objective prior to the determination of DNA contents
in trophoblast cells. A total of 150 presumptive normal and 150 presumptive t6mutant nuclei was measured. Machine units were converted to C values, i.e.
multiples of the haploid amount of DNA, by standardization with liver nuclei that
were identically fixed and stained. (A) Trophoblast nuclear DNA contents from
presumptive t6/t6 blastocysts; (B) trophoblast nuclear DNA contents from presumptive + // 6 and + / + blastocysts.
Studies on ^-mutant embryos
137
Table 2. Average nuclear numbers for normal and tG-mutant
trophectodermal vesicle outgrowths
Nuclear numbers
GD
Experimental
cross
Control
cross
7
8
9
10
11
9
13
13
12
10
10
12
12
12
11
Photographs of TVs from the experimental and control crosses which were used for
measurement of outgrowth areas (Fig. 3) were also used for nuclear counts. The experimental
cross data include determinations on both phenotypically normal and mutant embryos. Due
to the compactness of the outgrowths on EGD 7, some of the counts were underestimated,
explaining the increased nuclear numbers at subsequent stages. Nuclear numbers serve only
as an approximate indicator of cell numbers since in some cases it is not possible to distinguish
between two closely apposed cells and a binucleate cell (Sherman, 1975 a).
Table 3. DNA contents in outgrowths of normal and t6-mutant
blastocysts and trophectodermal vesicles
Blastocysts
Trophectodermal vesicles
A
1
Experimental cross
Experimental cross
A_
A
r
No. of structures
analyzed
No. of nuclei
measured
Average C value
% of nuclei
diploid
% of nuclei 8 C
or less
% of nuclei 32 C
or greater
Mutant
phenotype
Normal
phenotype
6
9
15
25
9
150
150
62
94
40
13-2
6
42-8
0
35-3
0
130
11
680
2
Mutant
phenotype
Normal
phenotype
Control
cross
Normal
phenotype
29
5
37
1
3
19
80
0
80
78
Values are taken from the data in Figs. 4 and 5 except for control cross TV nuclear values,
the distribution of which is not shown. DNA contents could not be obtained for all trophoblast nuclei since in some cases nuclei were overlapping, too close to each other to allow
separate measurement or, in the case of normal blastocysts, obliterated by ICM cells.
138
L. R. W U D L AND M. I. SHERMAN
256
Fig. 5. DNA contents of trophoblast cells from phenotypically normal and /6-mutant
trophectodermal vesicles. TVs from the experimental cross were cultured to the
12th EGD and treated as were blastocysts (see Methods and legend to Fig. 4).
Sixty-two (A) and ninety-four (B) trophoblast nuclei were measured from phenotypically mutant and normal TVs, respectively.
undergo about 2-5 more endoreduplicative cycles than mutant ones as reflected
by the average and largest DNA contents in the two groups. In other studies
(not shown), the DNA contents of the five largest trophoblast nuclei from each
of fifteen phenotypically normal and mutant bastocysts were measured. In this
case, the largest DNA content in a normal nucleus (655 C) was about three
endoreduplicative cycles beyond that found in a mutant nucleus (80 C).
We carried out similar studies with TVs from normal and mutant embryos on
the 12th EGD (we measured these one EGD later than blastocysts since TV
development is retarded by approximately a day relative to blastocysts). The
results were basically the same: normal trophoblast nuclei polyploidize almost
to the extent of their counterparts in blastocysts (Fig. 5, Table 3) and to degrees
similar to those in TVs from the control ( + / + x +/tG) cross (Table 3). Poly-
Studies on tG-mutant embryos
139
Fig. 6. Relationship between trophoblast nuclear diameters and DNA contents. At
the same time as DNA contents in 150 normal and 150 /"-mutant trophoblast nuclei
were determined (see Fig. 4), nuclear diameters were also measured. Regression lines
between these parameters were constructed by computer analysis. —, Nuclei from
phenotypically normal blastocyst outgrowths;
, nuclei from phenotypically
/"-mutant blastocyst outgrowths.
ploidization also takes place in most mutant TV nuclei, but not to the extent of
normals. In fact, more than one-third of mutant trophoblast nuclei have DNA
values at the level of 8 C or less, whereas almost all normal trophoblast nuclei,
both from experimental and control crosses, have DNA contents greater than
8 C. Conversely, four-fifths of the latter nuclei, but none of the former, have
DNA contents in the 32 C range or greater (Table 3).
We have examined more closely the relationship between nuclear size and
DNA content by constructing regression lines for these parameters for the 150
nuclei each from normal and mutant blastocysts analyzed in Fig. 4. The
regression lines for the normal and mutant nuclei, shown in Fig. 6, have similar,
but not identical, slopes. The difference in slopes indicates that, on average,
mutant nuclei of the same diameter as normal ones contain slightly less DNA,
suggesting, perhaps, that mutant nuclei flatten out more extensively on the culture
dish surface than do control nuclei.
The data presented so far do not indicate whether the differences in DNA
content between normal and mutant nuclei are due to continuing endoreduplication in the latter, although at a slower rate than in the former, to premature cessation of polyploidization in mutant nuclei after an initial period of
endoreduplication at a normal rate, or to a combination of decreased endoreduplication rates and premature cessation. We approached this question by
140
L. R. WUDL AND M. I. SHERMAN
I
T
I
I
If CD
10
1 1
5
JJL
0
10
10
5
0
£ 10
I
9
5
, It t Hal
0
10
8
5
0
10
7
5
0
T
20
II
40 0
Average nuclear diameter (/um)
II
20
1
40
Fig. 7. Average trophoblast nuclear diameters of trophectodermal vesicles derived
from control and experimental cross embryos. Diameters were determined for trophoblast nuclei in each of the TVs described in the legend to Fig. 3 on the 7th through
11th EGD. Each circle represents the average diameter of all the nuclei in an individually cultured TV. # , TVs from the control cross; O, TVs from the experimental
cross.
measuring nuclear diameters in trophoblast cells from normal and mutant TVs
on successive culture days and then estimating DNA contents from the regression lines in Fig. 6. Each circle in Fig. 7 represents the average nuclear diameter
of trophoblast cells in an individually cultured TV from the control or the
experimental cross. The circles in the left-hand panels represent values for
trophoblast nuclei in the control cross; there is a steady increase in the average
nuclear diameters with age. Similar analyses on the experimental cross (righthand panels) generally show a wider distribution of average nuclear diameters
than those observed in the control cross.
Studies on ^-mutant embryos
141
The average values in Fig. 7 have been further averaged for each day and the
results are plotted in Fig. 8. The average nuclear diameters increase in both
experimental (filled circles) and control (open circles) populations, although
at a somewhat lower rate in the former. When the experimental population is
separated into the high 60 % and low 40 % subpopulations for the reasons
described in previous sections, the result is an overlap between the high 60 %
subpopulation and the control cross population, indicating that nuclear diameters of trophoblast cells from presumptive + / + and + / / 6 TVs in both crosses
are similar. The low 40 % subpopulation shows a slight, but continuous,
increase in average nuclear diameters with time.
In Table 4 we have estimated DNA contents from nuclear diameters using
the appropriate regression lines in Fig. 6. As expected, the average estimated
DNA contents for normal nuclei in the experimental and control crosses rise in
parallel with time. The presumptive mutant nuclei show only a doubling in the
average estimated DNA contents over the 5-day period, but the rise is continuous.
Nucleic acid synthesis by ^-mutant and normal trophoblast cells
In order to obtain a clearer idea of the ability of ?6-mutant embryos to
endoreduplicate during the culture period, we incubated blastocysts or TVs in
[3H]thymidine for 24 h periods between the 6th and 11th EGD. We found that
from the earliest times investigated, i.e. shortly after the onset of outgrowth of
trophoblast cells, phenotypically mutant blastocysts or TVs incorporate the
isotope, but fewer of the trophoblast nuclei are labeled when compared to those
in normal outgrowths in the experimental or control cross (Table 5). As the
culture period proceeds, the proportion of labeled trophoblast nuclei in both
mutant and normal outgrowth falls, but the decline is more precipitous in the
mutant populations. Even so, between the 10th and 11th EGD in the latter case,
a few trophoblast nuclei can be found to incorporate [3H]thymidine. On the
other hand, the percentage of labeled trophoblast nuclei in normal blastocysts
levels off or even rises at this stage in culture as relatively small presumptive
secondary trophoblast cells (Sherman, 1975 b) can be seen to migrate out along
the culture dish from under some of the ICMs. These cells, which incorporate
label into their nuclei, are not seen in mutant blastocysts since the latter do not
have ICMs. The coincidental rise in labeled nuclei in phenotypically normal
TVs on EGD 10-5 is not due to the presence of secondary trophoblast cells, but
is probably an artifact related to the small number of nuclei measured at that
stage.
Since trophoblast nuclei have disparate sizes, shapes and distributions of
chromatin, we felt that grain counts would not provide a reliable indication of
labeling intensity. However, by visual inspection, it appeared as though the
grain density was consistently lower over mutant than over normal nuclei,
most obviously for the EGD 9-5 samples.
A further series of experiments was carried out to determine whether ?6IO
EMB 48
142
L. R. WUDL AND M. I. SHERMAN
40
^
-
20-
8
9
10
11
Hquivalent gestation clay
Fig. 8. Relationship between average trophoblast nuclear diameter and equivalent
gestation age for outgrowths of trophectodermal vesicles from the control and
experimental crosses. Each circle on these curves represents an average of the
averages illustrated in Fig. 7. O, Control cross; • , experimental cross. Also, the
lowest 40% of the experimental cross values (A) from Fig. 7 have been averaged
separately from the highest 60 % (A).
Table 4. Estimation of trophoblast nuclear ploidy values from
nuclear diameters
Experimental cross ( + /t6x +/7 e
Presumptive mutants
(low 40%)
A
I
EGD
8
9
Average
nuclear
diameter
.
\
Estimated
average
ploidy
(C)
Presumptive normals
(high 60 %)
Average
nuclear
diameter
(/tin)
Estimated
average
ploidy
(Q
150
21-7
180
28-9
19-2
34-3
10
19-8
34-9
11
Average nuclear diameter values are taken from the
made from the appropriate regression lines in Fig. 6.
Control cross
Average
nuclear
diameter
17
29
42
44
data in Fig.
Estimated
average
ploidy
(C)
210
15
34
30-7
42
34-3
52
37-9
8. Ploidy estimates are
—
—
53
113
177
146
107
—
—
77-4
59-3
48-0
20-5
6-5
410
370
401
503
345
209
199
162
86
No. of
nuclei
A_
63-2
61-6
42-9
16-6
119
46-9
27-1
25-3
7-0
^
Nuclei
labeled
(%)
Expt. 2
1
—
—
231
212
213
360
424
No. of
nuclei
98-7
97-2
80-3
45-6
65-8
—
—
—
663
743
471
614
447
107
61
121
41
No. of
nuclei
93-5
79-5
72-2
49-3
46-3
87-9
75-4
47-9
610
Nuclei
labeled
(%)
A
Nuclei
labeled
(%)
Expt . 2
A
1
Expt . 1
778
630
392
481
347
98-6
97-8
91 8
61-7
58-2
Expt . 1
\
^Nuclei
No. of
labeled
nuclei
(%)
<
Control cross
422
415
370
299
316
No. of
nuclei
A
98-6
97-8
941
62-9
45-6
Nuclei
labeled
(%)
Expt. 2
( + /+x +/n
A
Blastocysts or TVs were incubated with [3H]thymidine for 24 h periods and prepared for autoradiography as described in Methods.
EGDs are given in half days to reflect the fact that the radioisotope was administered on one day and the embryos were collected one day
later. In the experimental cross, structures were scored as phenotypically mutant or normal by their trophoblast nuclear and cell sizes before
being analyzed for the presence of grains. Only trophoblast cells were scored for the uptake of labeled thymidine; these could be distinguished by morphology from the smaller endoderm cells that migrate out from the ICM in some of the normal blastocysts. A nucleus was
considered to be labeled if the grain density was at least twice that of the surrounding cytoplasm. In general, background grain density was
very low and there was little question as to whether the nuclei were legitimately labeled. It is possible that the percentage of nuclei labeled in
blastocysts from the control cross drops less rapidly than that of phenotypically normal blastocysts in the experimental cross because the
former appear to be somewhat retarded compared to the latter at the time of collection from the uterus; this is also reflected by the difference
in /?-glucuronidase levels (see Fig. 1).
6-5
7-5
8-5
9-5
10-5
7-5
8-5
9-5
10-5
Blastocysts
TVs
EGD
Structures
,
f
A
Expt . 1
^
*Nuclei
labeled
No. of
nuclei
(%)
Normal phenotype
A
+ //')
Mutant phenotype
A
Experimental cross ( + /tGx
Table 5. Incorporation of [3H]thymidine by normal and t6-mutant blastocyst and trophectodermal vesicle outgrowths
<<_
sr-t-
o
<v
Co
144
L. R. WUDL AND M. I. SHERMAN
mutant trophoblast cells continued to incorporate [3H]uridine into RNA
throughout the culture period. Even when the labeled nucleoside is administered
to blastocysts for as little as 4 h, virtually all (> 90 %) trophoblast cells, whether
phenotypically normal or mutant, are labeled when studied by autoradiography.
Although grain densities appear by visual inspection to be lower over mutant
than over phenotypically normal trophoblast cells on EGD 8, gross differences
are not apparent either at earlier stages or beyond EGD 8, at which time the
grain density seems to diminish over both mutant and normal outgrowths.
t^-mutant: normal chimeric embryos
We generated chimeric blastocysts by fusing pairs of 8-cell experimental cross
embryos in order to investigate whether (1) t6/^ ICMs die in culture because
they have subnormal cell numbers (Sherman (1975#) has shown this to be the
case for miniblastocysts developing from disaggregrated blastomeres) and (2)
mutant trophoblast cells might polyploidize normally if in direct cell-cell contact
with normally replicating trophoblast cells.
When we inspected outgrowths developing from chimeric blastocysts on the
10th EGD, we found three types that could be distinguished by morphology
(Fig. 9): the first type, from presumptive normal: normal chimeras, looks like
a large, phenotypically wild-type embryo with a substantial ICM and many
giant trophoblast cells; the second type of outgrowth, presumably from mutant:
mutant embryos, contains only undersized, although healthy-looking, trophoblast cells, and little or no evidence of surviving ICM cells; finally, the putative
normal:mutant chimeras give rise to structures containing some ICM cells and
two discrete clusters of trophoblast cells, one of normal size, the other distinctly
undersized. The results indicate that t&/t6 ICMs are not rescued in vitro by being
combined in pairs, and that / 6 /7 6 trophoblast cells do not acquire a normal
phenotype by coming into contact with normal trophobtast cells.
Differentiation of'^-mutant trophoblast cells
Table 6 indicates that plasminogen activator activity can be detected in
blastocysts by the fibrin-agar overlay assay between the 6th and 7th EGD and
the enzyme continues to be secreted by all embryos, mutant and normal, up to
the 10th EGD. Since beyond that point plasminogen activator production is
more marked in parietal endoderm than trophoblast cells (Strickland et al.
1976) and only normal blastocysts would possess the former cell types, we
carried out similar studies on TVs, wherein both normal and mutant outgrowths
would contain only trophoblast. We found that similar numbers of outgrowths
from both the mutant and normal crosses produce plasminogen activator. As
reported earlier (Sherman et al. 1976), the amount of enzyme produced and
secreted by TVs drops beyond EGD 10, so that the size of the fibrinolysis zones,
and, eventually, the number of outgrowths secreting detectable amounts of
enzyme, decreases. There is a greater number of negatives in the EGD 12-5
Studies on tG-mutant embryos
Fig. 9. Outgrowths of experimental cross chimeric blastocysts. Structures were
photographed on the 10th EGD. (A), Presumptive normal:normal chimera; (B)
presumptive /6-mutant:/6-mutant chimera; (C) presumptive normal:f"-mutant
chimera. Scale marker (in C) = 50 /*m.
145
146
L. R. WUDL AND M. I. SHERMAN
Table 6. Plasminogen activator production by normal and t^-mutant
cultured blastocysts and trophectodermal vesicles
Control cross
( + / + x +/tG)
Experimental cross
(+/t6x+/t6)
A
A
t
Structures
EGD
Blastocysts
5-5
6-5
7-5
8-5
9-5
7-5
TVs
0
20
21
24
18
10
0
0
0
0
0
2
-
+
±
-
5
0
0
0
0
3
0
17
31
30
10
7
0
0
0
0
0
1
3
1
1
0
1
5
0
0
0
0
1
0
0
8-5
31
4
5
27
9-5
10-5
11-5
31
36
13
3
0
21
1
1
19
2
12-5
3
1
0
5
13
8
0
0
1
Blastocysts or TVs were prepared and tested for plasminogen activator production by the
fibrin-agar overlay procedure as described in Methods. EGDs are given in half days because
the assay period was 24 h; thus, for the EGD 5-5 samples, for example, blastocysts were
placed under the fibrin-agar overlay on the 5th EGD and scored for the presence of lysis zones
on the 6th EGD. Structures were scored + if a lysis zone was clearly visible surrounding the
entire outgrowth, ± if only a very faint or markedly assymetric lysis zone was present (see
Sherman et al. 1976), and — if no lysis zone was visible after the overlays were stained with
Coomassie brilliant blue.
10 5 -
50
100
T
500
1000
5000
Progesterone produced (pg)
Fig. 10. Progesterone production by individual blastocysts from the control and
experimental crosses. 3/?-HSD activities were estimated by the ability of 9th EGD
blastocysts to convert pregnenolone in the culture medium to progesterone (see
Methods). Prior to the collection of the culture medium for assay, blastocyst outgrowths in the experimental cross (A) were phenotyped: O, presumptive + /t6 or
+ / + 5 •> presumptive t6/tG. (B) blastocyst outgrowths from the control cross. Each
circle represents the amount of progesterone produced by a single blastocyst
outgrowth over a 24 h period.
Studies on ^-mutant embryos
147
sample in the experimental cross than in the control cross because, as we have
mentioned previously (Wudl et al. 1977), cell death is apparent by this time in
t6/tG TVs, and the remaining viable trophoblast cells in these structures presumably cannot produce sufficient amounts of plasminogen activator to be
detected by our assay.
We assayed blastocysts from control and experimental crosses for 3/?-HSD
activity by culturing them individually in the presence of pregnenolone (on the
9th EGD). The culture medium was collected and assayed for progesterone
production 24 h later. All of the outgrowths produce and secrete progesterone
(Fig. 10). However, the distribution of the amounts of progesterone detected is
very different in the two crosses, with the average progesterone content being
substantially higher in the experimental than in the control cross. This is mainly
because the media from phenotypically mutant blastocysts contain on average
much more progesterone than the media from normals (compare open and
filled circles in Fig. 10 A). This is explained by the fact that the mutant blastocysts have lost their lCMs by the time of the analysis: we have observed previously (Sherman & Atienza, 1975, 1977; Sherman, Atienza, Salomon & Wudl,
1977) that if ICMs are present, a substantial proportion of the progesterone
formed may be metabolized and so will not be detected by the selective radioimmunoassay procedure that is used.
DISCUSSION
Whereas the ICMs of putative t6/t6 blastocysts die at an early stage of
culture, trophoblast cells remain viable for several days thereafter, although
they are obviously affected by the mutation in that they fail to endoreduplicate
normally. It has been proposed that t mutations interfere with cell-cell organization or interactions (see Bennett, 1975). However, /6-mutant trophoblast cells
grow out in culture to give monolayers with a regular appearance; when compared to normals, mutant cells show no increased tendency either to migrate
away from, or pile up on, each other (Sherman & Wudl, 1977; Wudl etal. 1977).
In their gross morphology, presumptive t.6/ts trophoblast cells appear different
from normals only in that their nuclei do not become very large (see, for example,
Fig. 9). Although statistical analyses indicate that the nuclei of mutant trophoblast cells contain slightly less DNA per unit area than those of normals (Fig. 6),
this difference is not reflected by a visible alteration in nuclear morphology and
its significance is unclear.
Taken together, the data we have presented indicate that the difference in
nuclear size between presumptive tG/tG and normal trophoblast cells is related
primarily to differing DNA contents in the nuclei. Although other explanations
of the data in Tables 4 and 5 and Fig. 5 might not be formally ruled out, we
believe the most likely interpretation of these results is that polyploidization
takes place continuously in £6-mutant trophoblast nuclei but at a consistently
148
L. R. WUDL AND M. I. SHERMAN
slower rate than in + / + or + /tG trophoblast nuclei. Some presumptive tG/t6
trophoblast cells cease endoreduplication 1 or 2 days prior to normals, but others
continue to synthesize DNA for the same length of time as normals. Autoradiographic experiments designed to detect the incorporation of uridine into RNA
indicate that those trophoblast cells which have ceased replication, both mutant
and normal, nevertheless continue to synthesize RNA.
Since trophoblast cells in genotypically normal TVs polyploidize at almost
the same rate as those in blastocysts, it is unlikely that fc-mutant trophoblast cells
fail to polyploidize normally due to lack of interaction with 1CM cells. The
likelihood is further reduced by our observation that putative mutant trophoblast cells are retarded in their rate of endoreduplication even as part of a chimera
containing phenotypically normal trophoblast and ICM cells.
Despite its adverse effects upon polyploidization, the t6 mutation does not
prevent trophoblast cells from producing 3/?-HSD and plasminogen activator,
two specialized enzymes that are characteristic of trophoblast differentiation
(Chew & Sherman, 1976; Strickland et al. 1976). From previous observations,
we can state that these enzymes are first detectable in normal embryos at about
the time at which we can discern clear differences between normal and mutant
embryos (loss of ICM cells and subnormal levels of /?-glucuronidase activity).
Because we have not determined the time of transcription of the 3/?-HSD and
plasminogen activator genes, we do not know whether they were activated in
^-mutant cells prior to, during, or subsequent to interference with normal
development. We can, however, argue that synthesis of these differentiated gene
products is continued at substantial levels after the cells are otherwise affected
by the mutation. It is unlikely that J6-mutant trophoblast cells are merely
secreting plasminogen activator formed while they were still phenotypically
normal because Strickland et al. (1976) found that the amount of enzyme
present in blastocyst cells at any time is only a small fraction of the amount
being secreted. Furthermore, the levels of 3/?-HSD activity in cultured presumptive £6/76 embryos as reflected by progesterone production (1-2 pmoles/day)
compares favorably with those observed for wild-type embryos in which the
ICM had been eliminated by antimetabolite treatment (Sherman & Atienza,
1975). We conclude, therefore, that the tG mutation does not interfere directly
with known differentiative processes in trophoblast cells.
Since our observations render it unlikely that the primary action of the t6
mutation on trophoblast cells is interference with cell interactions or differentiation, we favor the view that the initial lesion is a metabolic one, that is, that
the + t 6 gene product(s) is directly involved in basic cellular metabolism (see
Hillman, 1975; Sherman & Wudl, 1977). Such a lesion could explain why ICM
cells, which might require intense metabolic activity for their rapid rates of cell
division, would succumb to the effects of the t6 mutation abruptly whereas nondividing trophoblast cells might be able to persist for a longer period of time
under suboptimal conditions. In fact, this is what happens when blastocysts are
Studies on ^-mutant embryos
149
cultured in the presence of appropriate doses of various antimetabolites (Rowinski, Solter & Koprowski, 1975; Sherman & Atienza, 1975; Pedersen & Spindle,
1976). Because replication is an energy-requiring process, it is not difficult to
visualize a drop in the rate of polyploidization of £6-mutant trophoblast cells
under conditions of metabolic insufficiency. We have found recently that certain
suboptimal culture conditions prevent expression of some trophoblast differentiation markers but not of others (M. H. Sellens, L. R. Wudl & M. I. Sherman,
unpublished observations); this could be analogous to the pronounced degree
of interference by the f6 mutation with trophoblast polyploidization relative to
3/?-HSD and plasminogen activator production.
Nadijcka & Hillman (1975) have reported that presumptive t6/tQ embryos
can be distinguished from normal embryos by ultrastructural analyses at substantially earlier stages than those in which we have been able to discriminate
between the two on the basis of /?-glucuronidase activities and death of ICM
cells. This would imply that ?6-mutant gene expression occurs at early stages of
development with relatively minor consequences, yet its effect becomes lethal,
and abruptly so in so far as the ICM is concerned, at a later period in embryogenesis. Such a phenomenon might be understood by taking into account the
observations that cleavage-stage embryos can develop to the expanded blastocyst stage in a medium containing only salts, glucose, pyruvate and albumin
(e.g. Biggers, Whitten & Whittingham, 1971), yet, for further development, a
complex mixture including amino acids, vitamins and serum factors is necessary
(e. g. Gwatkin, 1966). Thus, it appears as though the embryo is progressing
through a period of increased 'metabolic sophistication' during the phenocritical period for the t6 mutation, and a gene product which played only a
limited role just prior to this period could become crucial for normal cellular
metabolism within a very short time.
We have found the comparative study of normal and /6-mutant TVs to be
relatively simple because only a single cell type is involved, and advantageous
because it is possible to unambiguously phenotype homozygous f6-mutant cells
several days before their death. This advantage does not apply to later-acting t
mutations. For example, normal and mutant TV outgrowths from a (+ /tw5 x
+ /twS) cross are morphologically indistinguishable from each other as late as
the 11th EGD (unpublished observations). Therefore, the t* TV system might
prove to be particularly useful in our attempts to understand the mode of action
of this mutation. This in turn might help to clarify some of the mystery
surrounding T-complex mutations in general.
We are grateful to Ms Jill Lunn for statistical analyses and to Dr A. Weissbach for
comments on the manuscript.
150
L. R. WUDL AND M. I. SHERMAN
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{Received 17 April 1978, revised 11 July 1978)