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/ . Embryol. exp. Morpli., Vol. 15, 2, pp. 119-124, April 1966
With 2 plates
Printed in Great Britain
119
Oligosyndactylism, an early embryonic lethal
in the mouse
By PHEBE VAN VALEN 1
From the Department of Animal Genetics, University College London,
and the Department of Pharmacology, New York University
Medical Center
In heterozygous condition, the gene for oligosyndactylism (Os) in the mouse
(Mus musculus) causes syndactylism on all four feet and sometimes suppression
of digit 2. Os/Os homozygotes are inviable and die before the 12th day of
gestation (Griineberg, 1956, 1961). The present study was initiated to identify
the OsjOs homozygote and to establish the circumstances of its death.
METHODS
The stock of Os mice studied was maintained in the Department of Animal
Genetics, University College London. During the later part of the work, a
colony was established at New York University Medical Center from mice
kindly sent by Professor Griineberg. Embryos from Os/+ xOs/ + and from
Osj + x +1+ matings were examined between the 3rd and 8th day of gestation.
Age was determined by the standard vaginal plug method. The morning on
which a vaginal plug was found was taken as day 0. Capsules containing 7- and
8-day-old embryos were removed from the uterus before fixation. In 5- and
6-day pregnancies the entire uterus was fixed, but only the regions of decidual
swelling were sectioned. There was good agreement between the number of
implantations and the number of recent corpora lutea. In the studies of 3- and
4-day-old embryos the ovaries, oviducts and uterus were sectioned since there
was no external evidence of the location of the embryos. The ovaries were
examined immediately for young corpora lutea, which, unlike old ones, are a
rosy pink before fixation, to determine if these females were pregnant. This
procedure eliminated the time-consuming preparation of late pseudo-pregnant
uteri. Most of the tissues were fixed in Bouin's solution, embedded in paraffin
wax, serially sectioned at 7, 8 or 10 /i, and stained with Ehrlich's hematoxylin
and eosin. A number of embryos 6 days and older were fixed in Carnoy's fluid
1
Author's address: Department of Pharmacology, New York University Medical Center,
550 First Avenue, New York, N.Y. 10016, U.S.A.
120
P. VAN VALEN
and stained with Azure B Bromide. However, this method was found to be less
satisfactory for studying cellular detail.
RESULTS
Embryos from Osj + x Osj + matings were classified morphologically as
normal or abnormal (Table 1). On the 3rd day there was no detectable OsjOs
class. The abnormal embryos seen on that day in both types of mating were
undivided eggs or eggs which had undergone only two cleavages. A distinct
class of abnormal embryos was first seen on the 4th day. By the 5th day resorption of these embryos was evident and on the 6th and subsequent days only
necrotic implantation sites remained. With one possible exception, abnormal
Table 1. Classification of embryos from Os/+ x O s / + matings
(days)
No. of
litters
3
4
5
6
7
8
5
10
4
14
6
2
Age
Total no.
embryos Normals
34
78
29
89
44
16
30
53
16
70
30
11
Living
Os/Os
23(29%)
—
—
—
—
Dead,
resorbing
—
11(38%)
19(21%)
13 (30%)
5(31%)
Abnormal Mean
non Os/Os litter size
4
2
2
—
1
—
6-8
7-8
7-2
6-4
7-3
80
embryos of this type were absent in Osj + x + / + matings (Table 2) and they
were therefore assumed to represent the OsjOs class. Twenty-three out of 78
living 4-day embryos were identified as Os\Os, in reasonable agreement with the
expectation (xl = H ) . In 5- to 8-day litters, there were 127 normal living embryos and 48 sites of resorbed embryos presumed to be OsjOs, again in good
agreement with the 3:1 expectation (xl = 0-55). The living 4-day embryos and
the resorption sites thereafter thus account for the entire OsjOs class. A single
4-day embryo from an Osj + x + / + mating was classified as OsjOs before
referring to the genotypes of the parents. Although the preparation was poor,
three abnormal cells, similar to those in OsjOs embryos, were found. Presumably the misclassification of this embryo was due to an artifact of preparation, or may represent a natural phenocopy.
Early on the 4th day OsjOs embryos contained abnormal cells but gross
morphology was normal. Abnormals were found only in litters of embryos of
at least 64 cells; i.e. the 6th cleavage division was completed. During the 3rd
day, the embryos examined consisted of from 16 to 50 cells. To illustrate the
findings at the beginning of the 4th day a typical litter will be described. Eight
normal embryos of approximately 65-75 cells had an average number of four
mitoses per embryo. Four OsjOs embryos in this litter each consisted of about
70 cells, 8-13 of which were characteristically abnormal. Two abnormal cells
may be seen in an embryo shown in Plate 1, fig. A. The cytoplasm is pale, the
J. Embryol. exp. Morph., Vol. 15, Part 2
PLATE 1
Fig. A. Early 4-day Os/Os embryo, x 1400.
Fig. B. Mid 4-day normal embryo, x 1400.
Fig. C. Mid 4-day Os/Os embryo, litter-mate of embryo in fig. B (x 1400). Inserted detail of
a pair of abnormal cells (x 2700).
Fig. D. Same embryo as fig. C (x 1400). Inserted detail of a single abnormal cell (x 2700).
P. VAN VALEN
facing p. 120
/. Embryol. exp. Morph., Vol. 15, Part 2
PLATE 2
Fig. E. Late 4-day normal embryo, x 1700.
Fig. F. Late 4-day Os/Os embryo, litter-mate of embryo in fig. E. x 1700.
Fig. G. 5-day normal embryo, x 1850.
Fig. H. 5-day Os/Os resorption site, litter-mate of embryo in fig. G. x 1700.
P. VAN VALEN
facing p. 121
An embryonic lethal in mouse
121
chromatin fragmented and pycnotic. At this stage both normal and abnormal
embryos have settled into uterine crypts and the trophectoderm has begun to
make contact with the uterine epithelium. Primary invasive cells (Wilson,
1963 a) are present between the trophectoderm and the uterine epithelium in
both normal and OsjOs embryos although they are somewhat more obscure
in the latter.
T a b l e 2 . Classification
of embryos
Age
(days)
No. of
litters
Total no.
of embryos
Normals
3
4
5
6
7
3
4
4
9
5
28
31
19
67
38
27
30
19
65
34
1
from
Os/+ x + / +
Dead,
resorbing Abnormal
—
—
—
2
1
11
I
—
—
3
matings
Mean litter
size
9-3
7-8
4-8
7-8
7-6
Similar to Os/Os.
The OsjOs embryos show their most striking abnormalities by the middle of
the 4th day. Again, one litter will serve as an example of the findings at this
stage. The sections from a typical mid-4-day OsjOs embryo and a section from
a normal litter-mate are shown in Plate 1, figs. B, C and D. Many cells of the
abnormal embryos were pale, containing fragmented, pycnotic chromatin and
lacking nuclear membrane and nucleolus. The chromatin fragments resembled
disordered mitotic figures. These cells were distributed in the trophectoderm
as well as in the ectoderm and endoderm of the inner cell mass. Frequently,
though not always, the abnormal cells appeared in pairs both showing extremely
similar patterns of degeneration (Plate 1; contrast inserts in figs. C and D). The
two OsjOs embryos from this litter had reached the same developmental stage
as their normal litter-mates. In all, the endoderm had differentiated in the inner
cell mass and cytoplasm from trophectoderm cells had penetrated the uterine
epithelium preliminary to implantation. The two OsjOs embryos consisted of
approximately 120 and 105 cells, of which respectively 41 and 32 were abnormal;
their normal litter-mates ranged from 98 to 170 cells with an average of 4 cells
in mitosis. Since the chromatin fragments resembled mitotic figures, it is possible
that a few normal mitoses in OsjOs embryos were mistakenly classified as
abnormal cells. However, an embryo was not classified as OsjOs unless it had
many more 'mitotic' cells than its litter-mates and contained at least some
cells with highly fragmented chromatin. The abnormal embryos were not
difficult to identify.
By the end of the 4th day most of the cells in OsjOs embryos were abnormal
(Plate 2, fig. F). Most trophectoderm cells were normal but they were sparse
and difficult to distinguish from maternal cells. OsjOs embryos were retarded,
though typically, as in the embryo illustrated, some further enlargement of the
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P. VAN VALEN
inner cell mass had occurred. Comparison with a normal sib shows that the
uterine reaction to the embryo was normal in that the epithelium had sloughed
away in the area next to the trophectoderm (Plate 2, figs. E, F). During the
5th day resorption of Os/Os embryos had begun and only a few embryonic
cells remained, all of which were abnormal. However, the decidual reaction
around these embryos seemed normal (Plate 2, figs. G, H). After the 5th day
the implantation sites were rilled with maternal red blood cells, phagocytes and
a few pycnotic cells which may have been of embryonic origin, and the decidual
region was also becoming infiltrated with phagocytes.
DISCUSSION
Two important aspects of the OsjOs embryonic abnormalities are: the frequent proximity of pairs of cells in the same stage of degeneration, and the
resemblance of some of the chromatin fragments to mitotic figures. These
characteristics lead to the speculation that individual cells became abnormal
either during mitosis or just after separation of daughter cells so that return to
interphase is prevented. This possibility is supported by the finding that these
cells never contained nucleoli or a nuclear membrane as did the interphase
cells of the same embryo. Abnormal cells occurred in all regions of the embryo
and it is probable that the cells entered their last division in normal sequence.
Since no abnormal cells were seen before the 64-cell stage we can assume that
the cells became abnormal during the 7th or 8th divisions.
It is not surprising that the uterine reaction to the embryo was normal,
because the Os/Os embryos are morphologically normal when implantation is
initiated. It is known that injected tumour-cell suspensions will implant, causing
the uterine epithelium to slough away (Wilson, 19636). Also, trauma and introduction of inert substances, such as oil, into pseudo-pregnant uteri, will cause
decidual swelling (Finn & Hinchliffe, 1964).
There is insufficient evidence, as yet, to relate the abnormalities of the heterozygote and the lethality of the homozygote. The digital fusions in Osj + have
been traced to a reduction of the preaxial border of the foot plates (Griineberg,
1961), and histochemical studies of the preaxial region of the apical ectodermal
ridge indicate more pycnotic cells than normal (Milaire, 1962). One could
speculate that cell death in the heterozygote and homozygote may have a
common basis, but supporting evidence is lacking. Another pleiotropic effect
of the Osj + genotype, diabetes insipidus (Falconer, Latyszewski & Isaacson,
1964), also cannot be related to the cause of death of the homozygote at present.
In the house mouse only two other early lethals are known. These are yellow
(Ay) at the agouti and t12 at the T(brachyury) locus. The t12jtx2 embryos die as
morulae and do not implant (Smith, 1956). Unlike OsjOs, AV\AV embryos
have an extended lethal period; retardation, nuclear pycnosis and subsequent
death occurring before, during and after implantation. The degree of differentia-
An embryonic lethal in mouse
123
tion of giant cells varies and is apparently critical to the degree of implantation
and decidual proliferation (Robertson, 1942; Eaton & Green, 1962, 1963). A
more detailed comparison of t12, Av and Os homozygotes may provide valuable
information about the interaction between uterus and embryo necessary for
normal implantation and decidual proliferation.
Another recessive lethal mutation which is somewhat similar to OsjOs has
been described in the salamander Pleuwdeles waltlii by Gallien & Coullenot
(1964). The larva dies after hatching with many cells blocked in metaphase.
Recently, interest has been directed to the metabolic processes of early
development as an indication of genetic control of differentiation (Brown, 1964;
Gross, 1964), and early lethal mutations may provide valuable information in
this direction. For instance, studies of RNA synthesis in t12/t12 (Mintz, 1964a,
b, c) and in the lethal anucleolate mutant in Xenopus laevis (Brown & Gurdon,
1964) have demonstrated the importance of normal nucleolar function. It is
hoped that metabolic studies of OsjOs and AV\AV may ultimately reveal a biochemical basis for these developmental defects.
SUMMARY
1. The lethal homozygote of the gene for oligosyndactylism in the mouse
has been identified. OsjOs embryos are histologically normal until the beginning of the 4th day after fertilization (64-cell stage). At this time some cells,
often in pairs, become pale and the chromatin becomes fragmented and pycnotic. During the 5th day resorption begins.
2. It is concluded that the cells become abnormal during the 7th or 8th
cleavage division and are either unable to complete division or to re-enter
interphase.
3. Implantation and decidual proliferation appear to be normal.
4. This study provides no evidence of relationship between the abnormalities
of the heterozygote and lethality of the homozygote.
RESUME
'Oligosyndactylisme', une mutation Utah embryonnaireprecoce
chez la souris
1. On a identifie l'homozygote letal du gene de l'oligosyndactylisme chez la
souris. Les embryons OsjOs sont histologiquement normaux jusqu'au debut
du 4 e jour apres la fecondation (stade 64 blastomeres). A ce moment, quelques
cellules, souvent par paires, palissent et la chromatine se fragmente et devient
pycnotique. La resorption commence au cours du 5 e jour.
2. On conclut que les cellules deviennent anormales au cours de la 7 e ou 8 e
division de segmentation et sont incapables, ou bien d'achever cette division ou
bien de reprendre l'etat d'interphase.
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P. VAN VALEN
3. L'implantation et la proliferation deciduale apparaissent normales.
4. Ces recherches ne revelent pas l'existence d'un rapport entre les anomalies
de l'etat heterozygote et la letalite de Fetat homozygote.
The author wishes to thank Professor H. Gruneberg, who suggested this study and in whose
laboratory much of the work was done. His hospitality and encouragement are gratefully
acknowledged.
The author is also grateful to the Department of Pharmacology of N.Y.U. Medical Center
for providing the facilities for completing this work and especially to Mr George Pozsgay for
his enthusiastic assistance with the photography.
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{Manuscript received 24 September 1965)