/. Embryol. exp. Morph. Vol. 27, 1, pp. 147-154, 1972
Printed in Great Britain
147
Embryological effects of a minute deficiency in
linkage group II of the mouse
By G. R. DUNN 1
From The University of Tennessee-Oak Ridge Graduate School of
Biomedical Sciences and the Biology Division,
Oak Ridge National Laboratory
SUMMARY
A minute deficiency in linkage group II of the mouse, involving the short-ear locus and at
least one other functional unit, was found to be lethal by day 8 after coitus when homozygous.
The earliest time at which mutants could be detected was 7 days 16 h after coitus. Histologically, the mutant embryos showed overgrowth of ectoderm and trophoblastic giant cells.
The mesoderm was almost entirely lacking and the mutant embryos were smaller than their
normal litter-mates.
It was concluded that the mutant was first expressed morphologically late on day 7 after
coitus and that its primary effect was to stimulate growth of extra-embryonic ectoderm.
INTRODUCTION
In the course of the radiation experiments on mice conducted at this laboratory by W. L. Russell, over 200 mutations have been generated in the dilute
short-ear region of linkage group II. More than 100 of these mutations have
been used recently by L. B. Russell (1970) to construct a complementation map
of this region. An interesting result of this work is the discovery of complementing embryonic lethals. Among several independent short-ear lethals that yield
complex complementation patterns is the mutant 5RD300H, which L. B. Russell
(1971) concludes to be a minute deficiency involving the short-ear locus and at
least one other functional lethal unit, /4. The lethal mutant, 5RD300H (hereafter
designated sel), yields viable short-eared young when it is combined with several
independent dilute short-ear deficiencies that are preimplantation lethals (L. B.
Russell, unpublished) or with certain other short-ear lethal mutants (L. B.
Russell, 1971). It is therefore of interest to determine the time and mechanism of
action of se1. A preliminary report of this work has appeared earlier (Dunn,
1970).
1
Author's address: Institute of Animal Genetics, West Mains Road, Edinburgh EH9, U.K.
148
G. R. DUNN
MATERIALS AND METHODS
The lethal is maintained by intercrossing + sel/d+ mice (where d = dilute,
and + = wild type). The stock is tested periodically to assure that sex has not
been lost through recombination. All mice used in these experiments were
segregants of the above cross.
Embryos of known ages were obtained by caging males and females of the
desired genotypes and checking the females each morning for vaginal plugs.
The day a plug was found was designated day 0. Hour designations were only
approximate, being calculated from the middle of the dark period, which is
correlated to the time of ovulation (Braden, 1957).
Pregnant females were killed at the indicated times by cervical dislocation
and their uteri were removed. Nine days after coitus, embryos were scored as
(1) living if a heartbeat was seen, (2) having died after day 8 if the embryos had
completed rotation but had no heartbeat, or (3) having died before day 8 if an
egg cylinder stage or no embryo was found in the decidua. Eight days after
coitus, implants were scored as living if a well developed embryo was present, or
dead if only an egg cylinder or no embryo was found.
Histological preparations were made using the technique of Fekete, Bartholomew & Snell (1940) with minor modifications. The embryos were sectioned at
10 /-cm and stained with hematoxylin and eosin.
Table 1. Survival of implants to day 9 after coitus
No. of implants
No. of $$
examined
Mating*
l
d+_ d+
d+Xd+
Died
earlyt
Died
latej
Total
39(26-5)
39(26-5)
147
l
+ se +se
•x •
d+
d+
l
+ se d+
x
d+ d+
d+ +sel
Alive
20
69(47-0)§
20
105(70-5)
12(8-1)
32(21-4)
149
20
111(70-2)
7(4-4)
40(25-4)
158
20
116(73-0)
14(8-8)
29(18-2)
159
* Matings are indicated as $ x
X After day 8 after coitus.
t Before day 8 after coitus.
§ Numbers in parentheses are % of total.
RESULTS
Table 1 gives the results of the examination of uterine contents on day 9 after
coitus. In all four types of matings, embryonic death is high after day 8. Only
in the +seljd+ x +seljd+ mating is there a high death-rate prior to day 8.
Embryology of'se1
149
Table 2 shows the survival of implants to day 8 after coitus. Only one control
mating was examined since all three controls had given similar results on day 9.
The difference in survival between the two matings is highly significant, so it is
concluded that the mutants were dying prior to day 8.
Embryos ranging in age from 7 days 9 h to 8 days 9 h were examined histologically. Table 3 gives a summary of the classification of these embryos using the
criteria described below. The data conform reasonably well to the expected
3 normal/1 mutant ratio.
Table 2. Survival of implants to day 8 after coitus
No. of implants
No. of $?
examined
Mating*
+ se' + se'
—,— x——
a+
d+
-j— x-—
Alive
Dead
Total
20
120 (69-8)t
52(30-2)
172
20
132(86-8)
20(13-2)
152
2
X = 13-6; d.f. = 1;P < 001.
* Matings are indicated as $ x <$.
t Numbers in parentheses are % of total.
Table 3. Characterization of embryos
from —z— x —— matings
No. of implants
Age*
No. of $?
examined
7+9
7 + 16
7 + 20
8+0
8+9
4
4
2
1
3
r
Normal
Presumed
mutant
Abnormal!
Total
26
19
9
5
13
3
4
7
3
6
5
2
0
0
0
34
25
16
8
19
* Days + hours after coitus.
| These embryos had neither normal nor typical mutant morphology and are presumed
to be those which died due to other causes during this period.
At 7 days 9 h only three of 34 embryos examined were identified as mutants.
Furthermore, these three mutants were similar in developmental stage to
mutants examined at 7 days 16 h. Two of the 7 day 9 h mutants occurred in the
same litter, and their litter-mates were classified as normal early 7 day embryos.
This variability in developmental stage within the litter also occurred between
normal litter-mates at all ages. Therefore, variation within the litter in development of embryos is apparently a characteristic of the stock of mice used and
150
G. R. D U N N
Fig. 1. Sagittal section of presumed mutant (sel/sel) embryo at 7 days 16 h. This
embryo has been compressed during histological preparation. The extra-embryonic
ectoderm (a) has begun to proliferate abnormally and the endoderm (b) is folded.
xl25.
Fig. 2. Sagittal section of a normal ( + / + ) embryo at 7 days 16 h. Mesoderm (m) is
present in large amounts, whereas it is absent from mutant embryos, x 125.
Fig. 3. Sagittal section of presumed mutant embryo at 7 days 20 h. Ectodermal cells
(a) have filled all but the amniotic cavity of the embryo. Numerous giant cells (gc)
are present, x 125.
Embryology o/se 1
151
not a result of the mutation. Ignoring the three 'advanced' mutants, no embryo
was morphologically mutant at 7 days 9 h. Nor could size be used as a criterion
for recognizing mutants at this age. The orientation of 18 of the 34 embryos
Fig. 4. Presumed mutant embryo at 8 days 9 h. The embryo is a solid mass of ectodermal cells (a) surrounded by morphologically normal endoderm (b). Extensive
hemorrhage (h) is present. This embryo appears smaller than the other mutant
embryos because of the angle of sectioning, x 125.
Fig. 5. Section transverse to neural fold of a normal embryo at 8 days 9 h showing
neural groove (ng), epimyocardium (em), and blood islands (bi) in the yolk sac. x 125.
152
G.R.DUNN
examined at 7 days 9 h was such that measurements could be made of their
midsagittal length. These data do not show a bimodal distribution as would be
expected if the growth of the mutants were already affected at this age.
Mutant embryos could first be consistently identified at 7 days 16 h. The
mutants were smaller than the normal embryos and lacked mesoderm which is
normally present in a well defined layer by this age (compare Figs. 1 and 2).
There were also indications that the extra-embryonic ectoderm and giant cells
were beginning to proliferate abnormally.
The entire ectoplacental cavity and exocoelom were filled with ectoderm-like
cells by 7 days 20 h. The number of giant cells had increased considerably
(Fig. 3). Both embryonic and extra-embryonic ectodermal cells may have been
proliferating, since many mitotic figures were seen in both.
By 8 days 0 h only the most ventral portion of the amniotic cavity was not
filled with ectoderm-like cells. This latter cavity was completely filled with cells
by 8 days 9 h (Fig. 4); the mutants at this age were therefore masses of ectodermal cells surrounded by histologically normal endoderm. Mitotic figures
could still be seen in the ectodermal mass, indicating that the mutants were still
alive at this time. Their overall dimensions were approximately those of a normal embryo at early day 7. A portion of a normal embryo at 8 days 9 h is shown
in Fig. 5 to illustrate the typical developmental changes which occur during this
time period.
On the basis of these observations, the general characteristics of presumed
mutant embryos can be summarized as follows: (1) extensive proliferation of
extra-embryonic ectoderm, (2) nearly total absence of mesoderm, (3) folding of
the embryonic endoderm at the ventral end of the embryo, (4) increased numbers
of giant cells and extensive hemorrhage around the embryo, and (5) reduced
size relative to normal.
DISCUSSION
1
The primary defect of se is apparently in the extra-embryonic ectoderm
(which includes the trophectoderm). The genetic material missing is evidently
involved in the control of proliferation of this tissue. The massive hemorrhage
seen in deciduae containing mutant embryos is probably the result of overproliferation of giant cells, which have been shown to arise, at least in part, from
extra-embryonic ectoderm (Fawcett, Wislocki & Waldo, 1947). These cells may
be involved in the breakdown of the decidual tissue (Alden, 1948). Overproliferation of giant cells would then be expected to cause excessive destruction
of uterine tissue and extensive hemorrhage.
The virtual absence of mesoderm in the mutants is most simply explained as a
consequence of the overgrowth of the extra-embryonic ectoderm. Several
mechanisms for this phenomenon could be postulated, but the present data do
not permit any conclusions as to which is the most likely. Since se1 is a deficiency,
however, the possibility cannot be excluded that an additional functional unit
Embryology o/se 1
153
is involved which controls mesoderm formation. That the lack of mesoderm,
which presumably would be lethal if it were the only defect in an embryo, is not
due to the absence of the se locus in these mice is shown by the fact that the
combination of se1 with other deficiencies which overlap se1 only at se yields
viable young (L. B. Russell, 1971).
It does not seem likely that the morphology of the mutant embryo is due to
abnormal differentiation of mesoderm. This can be shown by comparing the
development of normal and mutant embryos. Normally, mesoderm first appears
between the ectoderm and endoderm at the junction of the embryonic and extraembryonic tissues; it then proliferates as a thin layer of cells between the endodermal and ectodermal layers. In the mutant embryos, however, there is proliferation of cells beginning in the region of the ectoplacental cone, an area
which normally never produces mesoderm. Furthermore, few cells are seen
between the embryonic ectoderm and endoderm of the mutant embryos. On the
basis of these facts, the interpretation of the development of mutants given here
seems to be the most likely, even though it is based on topographical characterization of the cell types involved rather than any intrinsic differences between
the germ layers.
The folding of the endoderm observed at all the stages examined is interpreted
to be the result of normal growth of this germ layer. Since little mesoderm is
formed, and since the size of the embryo is reduced, the normal mechanical
forces which would cause the endoderm to develop as a smooth layer are absent
and the excess endoderm therefore collapses upon itself. This conclusion is
further supported by the fact that both the distal and proximal endoderm
appear histologically normal and contain normal numbers of mitoses.
The time of expression of se1 has not been exactly determined. The typical
mutant syndrome is not seen before 7 days 16 h, with the exceptions noted
above. It does not seem likely that use of shorter and more precise time intervals
would offer any additional information. The variability in developmental stage
within litters, minimally estimated to be 6 h for these mice, sets a limit to
the precision which could be obtained with this type of analysis. It may be
concluded on the basis of the 7 day 9 h data, however, that se1 is not expressed
morphologically until the latter half of day 7 after coitus.
I wish to thank Dr L. B. Russell for supplying the initial stock of mice for these experiments
and for much helpful discussion. I also thank Mr N. L. A. Cacheiro for help in preparing the
figures. This research was supported by USPHS Predoctoral Fellowship 5-F01-45095-02 and
by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.
154
G.R.DUNN
REFERENCES
R. H. (1948). Implantation of the rat egg. III. Origin and development of primary
trophoblast giant cells. Am. J. Anat. 83, 143-181.
BRADEN, A. W. H. (1957). The relationship of the diurnal light cycle and the time of ovulation
in mice. /. exp. Zool. 34, 177-188.
DUNN, G. R. (1970). Embryological effect of a short-ear lethal allele in the mouse. Genetics,
Princeton 64, s 17—s 18.
FAWCETT, D. W., WISLOCKI, G. B. & WALDO, C. M. (1947). The development of the mouse
ova in the anterior chamber of the eye and in the abdominal cavity. Am. J. Anat. 81, 413—
443.
FEKETE, E., BARTHOLOMEW, O. & SNELL, G. D. (1940). A technique for the preparation of
sections of early mouse embryos. Anat. Rec. 76, 441-447.
RUSSELL, L. B. (1971). Definition of functional units in a small chromosomal segment of the
mouse and its use in interpreting the nature of radiation-induced mutations. Mutat. Res.
11, 107-123.
ALDEN,
{Manuscript received 18 March 1971)