/. Embryo/, exp. Morph. Vol. 36, 2, pp. 363-371, 1976
Printed in Great Britain
363
The developmental analysis of an embryonic lethal
(c6H) in the mouse
By SUSAN E. LEWIS 1 , HOWARD A. TURCHIN 1 ,
AND SALOME GLUECKSOHN-WAELSCH 1 .
From the Department of Genetics, Albert Einstein College of Medicine,
Bronx, New York
SUMMARY
A development study of the effects of the cm allele at the albino locus has identified the c0H
homozygote as an early postimplantation lethal. Homozygous c6H embryos can first be
recognized at 6|-7 days of gestation by abnormalities of the ectoplacental cone and parietal
endoderm. At 7£ days, mutant embryos appear severely retarded with obvious abnormalities
in all germ layers. All c6H homozygotes are dead and resorbed by 8 days of development. It is
proposed that the mutation interferes with the normal differentiation of the parietal endoderm,
ectoplacental cone and extra-embryonic ectoderm of the egg cylinder.
INTRODUCTION
Several radiation-induced mutations identified as lethal alleles at the albino
locus have been shown to be associated with a variety of biochemical, morphological and ultrastructural defects in homozygotes (Erickson, GluecksohnWaelsch & Cori, 1968; Thorndike et al. 1972; Trigg & Gluecksohn-Waelsch,
1973). Members of this allelic series show a complex pattern of complementation with each other (Gluecksohn-Waelsch, Schiffman, Thorndike & Cori,
1974). This report presents the results of a study of the cCH allele which acts as an
early prenatal lethal when homozygous. The stage of lethal action is identified,
and the abnormal morphogenesis of the mutant homozygote is described and
interpreted.
MATERIALS AND METHODS
Matings of mice heterozygous for cm and cch (chinchilla) served to produce
homozygous embryos for this study. The use of the c°h allele makes possible the
identification of cGHjccl1 heterozygotes since the albino allele interacts with
chinchilla to cause a noticeable dilution of the chinchilla pigmentation. The
following crosses were used:
cchjc6H x cchjc6H.
chi ci
1
for
CGH
homozygotes,
chi ch i f ° r controls.
Authors' address: Department of Genetics, Albert Einstein College of Medicine,
1300 Morris Park Avenue, Bronx, New York 10461, U.S.A.
364
S. E. LEWIS AND OTHERS
Table 1. Summary of results from dissection of embryos
Resorbed
Total
Age of
embryos
No.of
litters
cchlc6H
12-14 days
8-12 days
6 | - 8 days
6^—14 days
6
13
6
25
cchlc6H)
6 £-14 days
17
Matings
cchjc6H x
Total
cchjcch X
Normal Abnormal Resorbed
embryos embryos embryos
and
No. of Abnormal
embryos
(%)
34*
65
29
128
0
0
8
8
13
36
5
54
47
101
42
190
28
36
31
33
131
0
6
137
5
* All pigmented.
The classic methods of retrograde analysis (Gluecksohn-Schoenheimer,
1940; Smith, 1956) served to determine the stage at which the first symptoms
of developmental abnormalities of c6H homozygotes become noticeable.
For gross morphological analysis, the decidual swellings from uteri at
different stages between 6£ and 14 days were dissected and the developmental
state of the embryonic contents assessed. The study of embryos from control
litters served to establish the nature and incidence of non-specific abnormalities
unrelated to the homozygous lethal genotype.
Embryos from litters at 5 | - 7 | days of development were prepared for histological analysis by dissecting decidual swellings free from the uterine wall, and
fixing them intact in Bouin's fixative. They were embedded in paraffin, and
serial sections were cut at 8/*m and stained in Delafield's hemotoxylin and
Eosin Y.
RESULTS
Gross morphology
The gross morphology of 190 embryos from 25 cclljcm x ccllc6H matings and
137 embryos from 17 control litters was assessed at stages from 6|-14 days
after fertilization (Table 1).
Throughout this period, many more decidual swellings containing resorption
sites appeared in litters from matings of heterozygotes inter se than in those from
control matings. If the observed proportion of resorption sites in control litters
(5 %) is taken into account, the frequency of excess resorption in experimental
litters conforms well to the expected 25 % of c6H homozygotes (x2 = 0-62).
These data indicated that death and resorption of cGHjcm embryos occur during
the period of postimplantation development.
The results of dissections of litters from heterozygotes mated inter se were
divided into three age groups (Table 1).
Analysis of an embryonic lethal (c6H) in the mouse
365
Table 2. Summary of histological analysis o/c 6 H homozygotes
Embryos
Age of
Embryos
No. of
litters
Matin gs
cchjc6H x ccA/c6H
7J days
cchjcch x ccft/c<5tf
cch^c6H x cchjc6H
61 days
cchjcch x cchjc6H
cchjcch x cehjcch
5|-6 days
i
J
cchjc6H x cchjc6H
cchjcch x cchjc6H
Develop
Resorpmental
c6H Homo- Atypical tion
failure
0/
Normal
zygote
abnormal sites Total
/o
15
0
9
0
5
1
7
4
1
5
80
32
112
9
56
30
91
75
3
3
81
30
6
18
7
7
5
46
35
0
0
3
1
2
2
51
38
10
8
9
4
12
8-12 and 12-14 days
The 19 litters dissected at 8-12 and 12-14 days of development revealed no
traces of embryos or membranes in the resorption sites. Furthermore, the
presence of eye pigment in all embryos from six litters between 12-14 days of
gestation indicated that c6H homozygotes, expected to be albino, failed to
reach this stage; they are most likely included among the large proportion of
resorption sites. Death and total resorption of the c6H homozygous embryo
apparently occur prior to 8 days of development.
6\-8 days
At 6|-8 days of gestation some resorption sites were found to contain
remnants of retarded egg cylinders with abnormal morphology. These aberrant
egg cylinders were characterized by long translucent tails at their antimesometrial poles.
Histological analysis
7\ days
Serial sections of 80 embryos from nine litters of cchfc6H x cclllc6H matings and
32 embryos from four control litters were examined at 1\ days of development
(Table 2.).
As a result, the nine litters from matings of heterozygotes inter se were
found to include a significantly higher proportion (30%) of embryos with
developmental failure than did control litters (6%); the total frequency of
abnormals approximates the expected 25 % of cm homozygotes (Table 2).
Furthermore, as will be described, the abnormalities of most aberrant embryos
were unique and consistent and never occurred in embryos from control litters.
These abnormal embryos are therefore assumed to represent the class of c6H
homozygotes, and will be referred to as such in the subsequent description.
Although slight differences in developmental stage were found among the 30
24
EMB 36
366
S. E. LEWIS AND OTHERS
Analysis of an embryonic lethal (c6H) in the mouse
367
normal embryos from control litters as well as among the 56 normal embryos
from heterozygous matings, all had a primitive streak and a well defined
mesodermal layer (Fig. 1). Another group of 15 embryos from matings of
heterozygotes inter se had unique and consistent morphological abnormalities.
Compared with normal littermates, all these embryos were severely retarded in
growth (Fig. 2), and totally lacked a primitive streak or mesodermal layer.
A marked extension of the parietal yolk-sac endoderm into the decidual
tissue at the antimesometrial pole was a striking characteristic of abnormal
embryos and was observed best in longitudinal sections slightly peripheral to the
main axis of the embryos (Figs. 3, 4). A hyalin layer continuous with Reichert's
membrane surrounded the cells of the extended yolk-sac. In addition, in many
such embryos, the parietal yolk-sac layer along with Reichert's membrane
appeared to intervene abnormally between the ectoplacental cone and the
extra-embryonic ectoderm at the mesometrial pole.
The ectoplacental cone of c6H homozygous embryos was either very small or
totally absent. Although in normal embryos numerous mitoses were seen in the
proximal portions of the cone, none could be found in the equivalent regions of
cGH embryos. Occasionally, the cells in the area adjacent to the egg-cylinder were
tightly packed (Fig. 5), as is the case in normals (Fig. 1), while in other mutant
embryos the remnants of the cone consisted of a loose network of cells with
enlarged nuclei (Fig. 2), particularly where Reichert's membrane intervened
abnormally between the ectoplacental cone and the egg-cylinder. Such cells with
enlarged nuclei were never seen in this area of the ectoplacental cone in normal
embryos in this study.
Although there was a severe quantitative reduction of the embryonic ectoderm
of the mutant egg-cylinder, it was normally organized into a cohesive columnar
layer in contrast to the extra-embryonic ectoderm which was disorganized and
attenuated (Fig. 6). The pro-amniotic cavity was always present (Fig. 2) but the
ectoplacental cavity could usually not be found. No amniotic folds or other
FIGURES 1-5
Fig. 1. Normal embryo from cGU strain at 1\ days of development. Medial longitudinal section. Primitive streak (s), ectoplacental cone(c), parietal yolk-sac (jp). x 300.
Fig. 2. c6H homozygous embryo at 1\ days of development. Medial longitudinal
section. Note fold in mesometrial area of proximal endoderm (en), ectoplacental
cone cells with enlarged nuclei (c). There is a proamniotic cavity (a), but no ectoplacental cavity, x 300.
Fig. 3. Peripheral longitudinal section of the c6H homozygote in Fig. 2. Arrow
indicates the extension of parietal yolk-sac into the decidual tissue, x 300.
Fig. 4. Peripheral longitudinal section of c6H homozygous embryo at 1\ days of
development. Primary giant cells (g). Extent of parietal yolk-sac indicated by
arrow, x 300.
Fig. 5. cm homozygote at 1\ days of development. Medial longitudinal section.
Ectoplacental cone (c). x 300.
24-2
368
S. E. LEWIS AND OTHERS
«m
Analysis of an embryonic lethal (c6H) in the mouse
369
signs of differentiation of extra-embryonic membranes were ever seen in mutant
embryos.
The proximal endoderm of the c6H homozygous embryo often failed to show
the normal proximal distal differentiation into squamous and columnar
layers along the axis of the egg-cylinder. The endoderm cells were frequently
irregular in shape (Fig. 6), and the endoderm layer often folded along the
embryonic axis (Fig. 2) or thickened into a clump at the antimesometrial pole.
Normal primary giant cells could usually be distinguished at the antimesometrial pole of typically abnormal embryos (Fig. 4). Maternal decidual tissue
appeared to be normal in every respect.
6\ days
Among 112 embryos at 6-| days from matings of heterozygotes inter se only
six resembled cm homozygotes of later stages in every respect. In three additional
embryos typical defects of the ectoplacental cone (Fig. 7) and parietal endoderm
(Fig. 8) were associated with only slight retardation of growth and development.
Differentiation of the proximal endoderm and appearance of embryonic ectoderm were normal (Fig. 7), as were primary giant cells (Fig. 8). Embryos with
such characteristics may express the earliest indication of visible morphological
abnormalities in c6H homozygotes.
The total frequency of 6|-day embryos with developmental deficiencies in
litters from matings of heterozygotes inter se (18%) exceeded that in control
litters (7 %) but was significantly lower than at 1\ days of development. Furthermore, only 8% were uniformly abnormal and resembled the older c6H
homozygotes. Presumably, some homozygotes are indistinguishable from
normal at this stage.
5£ days
At even earlier stages no embryos were found with the characteristics of the
c6H homozygote at later stages among a total of 51 (Table 2). Furthermore, there
was no significant difference (x2 = 0-21) in the incidence of overall developmental failure between experimental and control litters at this stage (Table 2).
FIGURES 6-8
Fig. 6. Detail of Fig. 5 showing disintegrated extra-embryonic ectoderm (ex) adjacent
to well organized embryonic ectoderm {eni). Note beginning disorganization of
surrounding proximal endoderm {en), x 900.
Fig. 7. c6H homozygote at 6£ days of development. Note egg-cylinder with well
organized embryonic ectoderm (em), normal proximal-distal differentiation of
proximal endoderm (en) and abnormal ectoplacental cone (c). x 300.
Fig. 8. Detail of Fig. 7. Normal primary giant cells (g). Arrow indicates the hyalin
layer surrounding the extension of the parietal endoderm. x 900.
370
S. E. LEWIS AND OTHERS
DISCUSSION
The identification of a prenatally lethal phenotype in the mouse requires that
embryos with uniform abnormalities be found at a particular developmental
stage in expected frequencies. In this study, a group of embryos becomes
distinguishable from normal littermates at early egg cylinder stages, first by the
appearance of defects in extra-embryonic structures and subsequently by the
development of abnormalities of all germ layers. These embryos are classified
as cGH homozygotes; their death and resorption occur by the 8th day of
development.
In such mutants, ectoplacental cone as well as extra-embryonic ectoderm fail
to proliferate normally; these tissues show early signs of degeneration in contrast
to the embryonic ectoderm which appears structurally normal. The association
and similarity of defects of ectoplacental cone and extra-embryonic ectoderm
support the suggestion (Gardner & Johnson, 1975) that these structures have a
common origin in the trophoblast layer. However, not all trophoblast derivatives
are affected in the c6H homozygote. For example, primary giant cells appear
entirely normal. It has been proposed that in the course of differentiation a
stimulus from the inner cell mass induces and maintains proliferation of adjacent
cone cells while preventing giant cell transformation in the adjacent trophectoderm (Gardner, 1971; Gardner & Johnson, 1972). It is conceivable that in the
mutant, the failure of the ectoplacental cone and the extra-embryonic ectoderm
to continue normal proliferation might be due to a defect in the interaction
between inner cell mass and trophectoderm. This is also supported by the total
absence of mitoses and the observation of cells with enlarged nuclei in the
mutant ectoplacental cone adjacent to the egg-cylinder, indicating possible giant
cell transformation.
The unique and striking abnormality of the parietal yolk-sac layer which
invades the maternal tissue in spear-like fashion is another prominent characteristic of the c6H homozygote. Overgrowth of this layer has been reported in
embryos homozygous for tw5, a lethal recessive allele at the T-locus (Bennett &
Dunn, 1958) where, however, the excess cells accumulate in the yolk-sac cavity.
Thus, the invasiveness of the parietal endoderm in cm homozygotes is not likely to
be merely the result of uncontrolled proliferation; it might be caused by changes
in adhesive properties of these cells or of Reichert's membrane which is secreted
by them (Pierce, Midgely, Sri Ram & Feldman, 1962).
Reduction of embryonic ectoderm growth as well as failure of primitive
streak formation are probably secondary abnormalities. The atypical thickening
and folding of the proximal endoderm of the c6H homozygote is also likely to be
secondary; it is reminiscent of the disorganization of this layer observed in
other egg-cylinder lethals, e.g. t° (Gluecksohn-Schoenheimer, 1940), tu'5 (Bennett
and Dunn, 1958), Bid (Vankin 1956) and se1 (Dunn, 1972). All these mutants
share the symptom of reduced growth, indicating a possible relationship between
Analysis of an embryonic lethal (c6H) in the mouse
371
growth of the egg-cylinder and normal differentiation of the proximal endoderm.
However, not all of the various disturbances of differentiation in the c0H
homozygote are necessarily causally related to each other. They might have
independent causes, particularly since the lethal albino alleles appear to be
deletions (Gluecksohn-Waelsch et al. 1974) covering more than one genetic
locus.
This study was supported by grants from the National Institutes of Health, HD-00193,
GM-00110, GM-19100 and by a grant from the American Cancer Society, VC-64.
From a thesis submitted by S.E.L. in partial fulfillment of the requirements for the degree of
Doctor of Philosophy from the Sue Golding Graduate Division of Medical Sciences, Albert
Einstein College of Medicine, Yeshiva University.
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{Received 5 April 1976)