/ . Embryol. exp. Morph. Vol. 36, 2, pp. 373-381, 1976
Printed in Great Britain
373
Embryological study of a Tjt locus mutation (tw73)
affecting trophectoderm development
By MARTHA SPIEGELMAN, 1 KAREN ARTZT, 1
AND DOROTHEA BENNETT 1
From the Department of Anatomy
Cornell University Medical College
SUMMARY
Mouse embryos homozygous for the recessive lethal mutation tw73 show specific defects
in trophectoderm shortly after implantation. The trophectoderm and ectoplacental cone fail
to form the usual close association with the uterine decidua, and proliferation is markedly
reduced. The embryo proper ceases to develop beyond the two-layered stage and degenerates
and dies within 5 days of implantation.
INTRODUCTION
Five different types of recessive lethal f-mutations in the mouse have so far
been described that affect discrete steps in development by impairing the
differentiation of particular embryonic cell types. The effect of each different
type of mutation can be imputed specifically to interference with progressive
changes that normally occur at important transitional points. The mutant
genes that have been described until now have all interfered with the diiferentiation of cell types in the embryo proper (see Bennett, 1975, for review). We
describe here a Mnutation, tw7S, that appears to affect the trophectoderm, one
of the two derivatives of the earliest division of the ectoderm. The origin of tw73
in a feral population in Denmark has already been described (Dunn & Bennett,
1971), as have its genetic characteristics (Dunn, Bennett & Cookingham, 1973).
MATERIALS AND METHODS
Embryos were obtained from matings between mice heterozygous for tw73
at 6, 7, and 8 days post-copulation. They were fixed intact within their capsules
in Bouin's fluid for 12-24 h, embedded in paraffin, sectioned serially at 6-10 /*m,
and stained with hematoxylin and eosin. Nominal age of embryos was based on
vaginal plug and was more precisely determined by comparing histological
sections of normal littermates to Sobotta's figures (1911).
1
Authors' address: The Sloan-Kettering Institute for Cancer Research 410 East 68 Street
New York, New York 10021, U.S.A.
374
M. SPIEGELMAN, K. ARTZT, AND D. BENNETT
Table 1. Classification of embryos obtained from matings
between heterozygotes for tw73
Gestational
age of
embryos*
(days)
7
6
Number
of litters
Total
embryos
Normal
embryos
5
4
12
43
29
99
21
22
69
Embryos
Classified
as
tw73\tw73
16
5
24
Resorbed
embryos
6
2
6
Total %
abnormal
51%
24%
30%
* Normal embryos at 6 days are two-layered egg-cylinders; at 7 days primitive streak and
mesoderm formation are occurring; at 8 days head folds and occasionally somites are present.
RESULTS
Table 1 gives the classification, based on histological appearance, of embryos
obtained from matings between ?w73-heterozygotes. Because of the unusually
high transmission of lethal Mnutations by males, 0-95 in the case oitw73 (Bennett,
1975), the expected proportion of homozygous embryos is 47-5 %. At 8 days the
percentage classified as homozygotes is very close to the expected. At 7 days,
although the sample size is small, the proportion does differ (P < 0-02) significantly from expectation, and at 6 days we classify far fewer (P < 0-0001)
embryos as homozygotes than we should expect. This suggests, as pointed out
below, that not all members of the genetically abnormal class can be distinguished before 8 days.
An abnormal class of embryos, putative roW-homozygotes, can first be recognized at 6 days of development when normal litter-mates are double-layered,
elongate egg cylinders. The mutants are also egg-cylinders but they are smaller,
FIGURES
1-4
Fig. 1. Longitudinal section through 6-day normal embryo at elongating eggcylinder stage. The ectoplacental cone (epc) appears well embedded in the decidua.
The yolk sac, consisting of trophectoderm and parietal endoderm, is in close contact
with the decidua. E, ectoderm; TV, endoderm. x 220.
Fig. 2. Longitudinal section through 6-day mutant embryo which is a two-layered
egg-cylinder but is not elongated. An ectoplacental cone is not present. The trophectoderm is not in close contact with the decidua. E, ectoderm; TV, endoderm. x 220.
Fig. 3. Slightly oblique section through 7-day normal embryo at the primitive
streak stage. The parietal endoderm (P) is closely adherent to trophectoderm
cells (T) which appear to be attached to the decidua. E, ectoderm; TV, endoderm;
M, mesoderm; arrow, giant cell, x 220.
Fig. 4. Transverse section through 7-day mutant embryo. The embryo is still at the
two-layered stage. The decidua is not invaded by trophectoderm but instead large
spaces are present between the parietal endoderm (P) and the maternal tissue. Some
groups of small cells (T) and some multinucleate cells (arrow) may be derived from
trophectoderm but there has not been a transition to giant cells nor an attachment
to the decidua. E, ectoderm; TV, endoderm. x 220.
Tjt mutations affecting trophectoderm
#
375
376
M. S P I E G E L M A N ,
Κ.
ARTZT,
AND
D.
BENNETT
Tjt mutations affecting trophectoderm
311
with crowded cells that occasionally contain dense granules; the endoderm cells
are not flattened, and are sometimes vacuolated. These embryos also seem poorly
implanted; the trophectoderm is often not in contact with the uterine tissue. The
ectoplacental cone has not usually formed in the mutant embryos although in
normal littermates it is large and embedded in the uterine stroma (Figs. 1, 2). It
is not always possible to distinguish arrested ^7iJ-homoz;ygotes from occasional
embryos that are lagging slightly in rate of development.
By 7 days, however, when normal littermates are at the primitive streak stage,
the ^-homozygotes are distinctive. The embryo proper is arrested as an eggcylinder, with no evidence of primitive streak or mesoderm formation. Cells
are crowded and the two-layered organization of the embryo is sometimes
obscured. Furthermore, the yolk sac and ectoplacental cone are markedly abnormal, and these embryos have obviously failed to become properly implanted
(Figs. 3, 4).
In normal embryos the trophectoderm has proliferated and thickened into a
shell of large basophilic cells embedded in the decidua, and has started giant
cell transformation. This differentiation has not occurred in J^-homozygotes
(Figs. 3-6), where the trophectoderm consists of groups of small cells scattered
among the cells of maternal origin, and giant cells are rare.
In normal embryos Reichert's membrane seems to form a bond between the
two cellular layers of the yolk sac, and thus the endoderm becomes stretched
into a flat layer as the trophectoderm expands deeper into the decidua (Figs.
3, 5). The parietal endoderm does not become stretched in the mutant but instead remains close to the embryo. The yolk-sac cavity is therefore small, and
large spaces are present between the parietal endoderm and uterine tissues
(Figs. 4,6). Reichert's membrane is scanty in the mutant and is sometimes difficult
to see as a distinct eosinophilic layer. The ectoplacental cone is much smaller
than normal and has not penetrated far into the decidua. Giant cells are also
deficient (Figs. 7, 8).
Thus in r^-homozygotes the trophectoderm seems not to form sufficient
numbers of viable giant cells, nor to transform into the invasive and adhesive
tissue necessary for implantation.
FIGURES
5-8
Fig. 5. Trophectoderm of the 7-day normal embryo forms a shell of cells (T) embedded in the decidua. Several giant cells (arrow) are present. P, parietal endoderm.
x350.
Fig. 6. Trophectoderm of the 7-day mutant embryo has not formed an invasive shell
of cells and giant cells are not present. Clusters of small cells (T) may be of trophectoderm origin. P, parietal endoderm. x 350.
Fig. 7. Ectoplacental cone (epc) of 7-day normal embryo is large and compact.
Many maternal blood cells (be) are located at the margin of the decidua but no large
spaces separate the embryonic and maternal tissues. Arrow, giant cell, x 220.
Fig. 8. Ectoplacental cone (epc) of 7-day mutant embryo is fairly large and compact but large spaces separate the embryonic and maternal tissues, x 220.
378
M. SPIEGELMAN, K. ARTZT, AND D. BENNETT
Tjt mutations affecting trophectoderm
379
A specific defect in the trophectoderm cells is even more obvious at 8 days.
Normal embryos by this time often have several pairs of somites (Fig. 9).
Extra-embryonic structures, including the amnion, chorion, and allantois, have
formed in addition to a further expansion and differentiation of the yolk sac.
The ^ 5 -homozygotes, on the other hand, remain arrested as egg-cylinders,
mesoderm has not formed nor is there any sign of new extra-embryonic membranes. Nevertheless, the embryo proper is without obvious specific abnormalities although it is retarded (Fig. 10).
The trophectoderm contribution in normal embryos forms a complex network of large cells with vascular channels continuous with those of the uterine
tissue. Many maternal red blood cells and a few lymphocytes are present in these
trophectoderm-lined spaces but these maternal cells very rarely advance
beyond the trophectoderm network; in fact, the large trophectoderm cells
actively engulf maternal cells (Fig. 11).
In contrast, the trophectoderm of ^"-homozygotes is absent or so atypical
as to be unrecognizable. Neither mural trophectoderm nor ectoplacental cone
are securely attached to the decidua, and large spaces separate the embryo from
the decidua. A few clusters of small cells irregularly disposed outside the parietal
endoderm may be remnants of the original trophectoderm, but these isolated
cell groups in no way resemble the reticular shell of large cells comprising the
normal trophectoderm (Fig. 12); many of these cells are pycnotic. Very few
giant cells are seen and these are not in contact with the maternal tissues.
A striking feature in mutant embryos is the presence of numerous lymphocytes and polymorphonuclear leukocytes in the space separating the embryo
and the uterine tissue. This suggests that trophectoderm of the normal embryo
may shield it from maternal cells. In the mutant the parietal endoderm appears
to serve as a barrier between migrant maternal cells and the embryo proper. In
FIGURES
9-12
Fig. 9. Transverse section through 8-day normal embryo at the head fold-early
somite stage. The trophectoderm (T) constitutes a reticular shell of large basophilic
cells deeply embedded in the decidua. P, parietal endoderm; R, Reichert's membrane; E, ectoderm; N, endoderm; M, mesoderm. x220.
Fig. 10. Transverse section through 8-day mutant embryo which remains arrested
at the two-layered stage. An invasive trophectoderm is not present but some small
cells (T) in the large spaces separating the embryonic and maternal tissues are
probably of trophectoderm origin. P, parietal endoderm; E, ectoderm; N, endoderm.
x220.
Fig. 11. The trophectoderm of the 8-day normal embryo forms a network of large
phagocytic cells (T) which engulf maternal red blood cells (arrow). P, parietal
endoderm; R, Reichert's membrane, x 350.
Fig. 12. The trophectoderm of the 8-day mutant embryo consists of small cells (T)
and some larger pycnotic cells (T). A reticular shell in close contact with the decidua
has not formed. Many maternal red blood cells and lymphocytes are present in the
large spaces between the embryonic and maternal tissues. P, parietal endoderm,
x35O.
380
M. SPIEGELMAN, K. ARTZT, AND D. BENNETT
the ectoplacental cone, where parietal endoderm is not an intervening layer,
maternal lymphocytes and polymorphonuclear leukocytes are abundantly
mingled with the cells of embryonic origin.
DISCUSSION
w73
The haplotype t
defines a new complementation group for recessive lethal
mutations at the T\t locus. Like mutations in the five other complementation
groups that have so far been described (Bennett, 1975), the primary homozygous
effects of tw73 in embryos are limited to ectodermal derivatives. UnliJce these
other mutations, however, which all affect rather circumscribed varieties of
cells within the embryo proper, tw73 seems to produce its first detectable abnormalities in an extraembryonic derivative, the trophectoderm. The subsequent
abnormalities that appear in the embryo itself can most simply be attributed to
secondary degenerative phenomena that are consequences of the defective
trophectoderm. Since it has been shown (Gardner & Papaioannou, 1975, for
review), however, that the inner cell mass of the embryo produces an inductive
stimulus required for the proliferation of trophectoderm, it is not impossible
that morphologically undetectable abnormalities in the embryonic ectoderm are
responsible for the failure of trophectoderm development.
Detailed fine structural studies have been made of homozygotes for two different T\t locus mutations, during stages in development when abnormalities
are just becoming evident. In t9/?9 homozygotes, for example, cells of mesodermal nature emerging from the primitive streak fail to recognize and/or interact
with one another, either morphologically or functionally (Spiegelman & Bennett,
1974). In T\T homozygotes, on the other hand, cells of the neural tube, somites
and notochord all show extensive but inappropriate interactions with one
another (Spiegelman, 1976). Thus, in each of these cases the morphological
abnormalities in homozygotes appear to result from deranged cell-cell recognition or interaction. Even without an analysis of fine structure, it seems evident
that the same kind of failure is responsible for the defects seen in t7Sw homozygotes. In these embryos, the response of trophectoderm to the maternal tissue
appears to be defective and results in ineffective nidation.
Between 6 and 8 days after fertilization, the mutants show increasingly
severe defects of trophectoderm and its derivatives as compared to the extensive
invasion and expansion of normal trophectoderm. On the other hand, the embryo
itself merely remains arrested; although it does not show any signs of progressive development or growth during this period, neither does it appear to deteriorate. This observation suggests that although further enlargement of the embryo
may depend on some nutritive function of the trophectoderm, the relatively
simple two-layered egg cylinder is maintained even without an effective trophectoderm.
There is also an extensive accumulation of lymphocytes and polymorpho-
Tjt mutations affecting trophectoderm
381
nuclear leukocytes in the implantation site at this time. Although the accumulation of these cells is a normal accompaniment of embryonic resorption, in this
case they begin to congregate in large numbers at least two days before embryonic death. This fact may suggest that a maternal immune response against
the embryo is being mounted in the absence of effective trophectoderm, which
may normally mask the antigenicity of the embryo (Billington & Jenkinson,
1975).
This work was supported by ERDA Contract No. E (11-1) 2497 and NSF Grant No.
BMS 75-16354.
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{Received 5 January 1976, revised 5 May 1976)
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