/. Embryol. exp. Morph. Vol. 52,pp. 115-125, 1979
Printed in Great Britain © Company of Biologists Limited 1979
In vivo and in vitro studies on the early embryonic
lethal oligosyndactylism (Os) in the mouse
By HUGH F. PATERSON 1
From the MRC Mammalian Development Unit, Wolfson House, London.
SUMMARY
The development of mouse embryos homozygous for oligosyndactylism (Os) is arrested
during implantation. Histological investigations confirm a previous report that cells become
blocked in mitosis, and air-dried spreads of the mutant embryos reveal that large numbers of
cells accumulate in metaphase. Trophoblastic giant cells appear unaffected by the action of
the mutant gene both in utero and during culture over the lethal phase. It is proposed that
the form of endoreduplication undergone by giant cells renders them refractory to the
metaphase block.
INTRODUCTION
The mutation oligosyndactylism (Os) causes fusion of the second and third
digits in all four feet of mice heterozygous for the allele (Griineberg, 1956,1961).
Anomalies of muscular anatomy in the limbs (Kadam, 1962) and a form of
diabetes insipidus (Falconer, Latyszewski & Isaacson, 1964) also usually
accompany the mutation, with high renal output and inability to concentrate
urine arising from a paucity of nephrons in the kidneys (Stewart & Stewart,
1969).
No satisfactory explanation, however, has been advanced to link these effects
with the embryonic lethality which Os causes in the homozygous condition and
which is the subject of this study. The time of death of Os/Os embryos in utero
has been determined in an histological investigation by Van Valen (1966), who
found that abnormalities first occur on day 5 post coitum, leading to death
during the following 24 h with subsequent resorption. Affected blastocysts were
found to possess increasing numbers of pale-staining cells, lacking a nuclear
membrane and nucleolus, and in which the chromatin lay scattered and fragmented in the cytoplasm. The frequent occurrence of these cells in pairs together
with the general resemblance of the chromatin to mitotic figures, led the author
to postulate that the mutation interferes with cell division. It was suggested that
cells undergoing the seventh or eighth cleavage are either unable to complete
1
Author's Address: Department of Viral Oncology, The Imperial Cancer Research Fund,
Lincoln's Inn Fields, London WC2A 3PX. U.K.
116
H . F.
PATERSON
division or cannot successfully re-enter interphase, with resultant cytostasis
and developmental arrest.
The purpose of this study was to confirm Van Valen's findings and to examine
in more detail the nature of the abnormalities described. To this end, the results
of an histological study of Os/Os embryos in utero, together with an examina­
tion of chromosome morphology in air-dried spreads of the affected cells, are
presented here. These are further supported by observations of the behaviour of
the mutant embryos when cultured over the lethal phase.
MATERIALS A N D
Experimental
METHODS
animals
The adult mice used in this study were F hybrids obtained from matings
between males heterozygous for oligosyndactylism (Os/ + ) derived from linkage
stocks at the Institute of Animal Genetics, Edinburgh, and wild-type females
( + / + ) of the highly fertile CD-I strain (Charles River). This cross was adopted
as a rapid method of producing stocks of experimental animals.
Matings between F mice heterozygous for the Os allele (Os/+ xOs/ + )
were employed to produce litters containing homozygous mutant embryos.
Control litters were obtained by mating wild-type males with heterozygous
females (Os/+ x + / + ), thereby ensuring that any unforseen effects of the
mutation on the maternal reproductive system or on the cytoplasm of the ova
would be equalized in both groups.
Copulation was assumed to have occurred at the mid-point of the dark period
preceding discovery of a vaginal plug. For convenience of timing, animals were
maintained in an artificially reversed diurnal light cycle.
x
x
Histological
procedures
Air-dried spreads of whole embryos were prepared according to an adaptation
of the method described by Evans, Burtenshaw & Ford, (1972). Blastocysts
were flushed from uteri at approximately 115 h post coitum, this being the latest
time at which reasonably intact embryos could be obtained, although yield was
often low. These were incubated for 15 min at 37 °C in hypotonic solution
(0-8 % sodium citrate in H O ) , followed by a 30 min fixation in three parts
ethanol and one part glacial acetic acid. Each embryo was then transferred in a
minimum volume of fixative onto a clean microscope slide, and a small drop of
6 0 % acetic acid added immediately. As soon as the embryo started to disag­
gregate, the drop was drawn across the slide with the tip of a micropipette,
leaving a trail of ruptured cells adhering to the surface. Preparations were
allowed to dry for 30 min at room temperature before staining with Geimsa or
Aceto-orcein.
a
Studies on oligosyndactylism (Os) in mouse
117
Culture
Blastocysts at approximately 95 h post coitum were flushed from uteri with
PBI medium (Whittingham & Wales, 1969). Zonae pellucidae were removed
at this stage by gently shearing-offwith with a small-bore micropipette. Embryos
were washed and transferred into individual droplets of Eagle's Minimal
Essential Medium supplemented with 2 0 % foetal calf serum, 1-6 mM glutamine
and 100 u n i t s / m l penicillin. Droplets were maintained under paraffin oil in
Falcon dishes at 36-5 °C in an atmosphere of 5 % C 0 in air.
The progress of individual embryos was recorded daily following examination
under phase-contrast optics. After 72 h of culture, the number of outgrowing
trophoblast cells (all sizes) in each embryo was determined, and the area of
outgrowth measured with a calibrated micrometer grid.
2
RESULTS
Histological
analysis
5 days p.c. (Data summarized in Table 1)
A total of 45 embryos in six uteri from Os/ + x Os/ + matings were sectioned
at this stage. The majority were present as late blastocysts/early egg-cylinders
(Fig. 1C). Twelve embryos (26-7%), however, were clearly abnormal (Fig.
la, b). These were presumed to be Os/Os on account of the overwhelming
similarity to the mutant homozygotes previously described by Van Valen (1966),
and of the excellent statistical agreement with the Mendelian expectation of
2 5 % . Gross morphology was usually somewhat mis-shapen, and frequently
the blastocoele had collapsed so that the trophectoderm was folded up around
the inner cell mass. Nevertheless, in most cases signs of attachment and initial
implantation could clearly be seen. On average, approximately 4 0 % of the cells
in these embryos were in a state resembling metaphase, and they were conse­
quently quite distinct from their litter-mates which typically displayed only one
tenth of this proportion of metaphases (Table 1).
Cell number was estimated according to the formula of Abercrombie (1946)
for nucleated cells, and by direct counting for mitotic cells. The mean number of
mitotic-type cells per embryo was thus calculated as 38-0 ( ± 2-8) for Os homo­
zygotes and as 5-6 (±0-7) for litter-mates, while mean total cell number per
embryo was estimated as 96-0 (±9-5) and 130-0 ( ± 6 - 7 ) for each group re­
spectively.
Student's /-test confirms that cell number in the Os homozygotes is signi­
ficantly lower than in litter-mates (P < 0-01).
6 days p.c.
Normal embryos at this time possessed well-formed egg cylinders. Attach­
ment was now complete, and each implantation site was surrounded by a
118
H. F. PATERSON
Studies on oligosyndactylism (Os) in mouse
119
Table 1. Day-5 embryos from O s / + x O s / + matings sectioned in utero
Class of embryo
Presumptive Os/ Os
Litter-mates
No of % total
Mean no.*
embryos embryos Mitotic-type cells
12
33
26-7
73-3
38O(±2-8)t
5-6 (±0-7)
Mean total
Cell no.
Mitotic index
96-0 (±9-5)
1300 (±6-7)
39-6%
4-2%
* Includes all cells in a state resembling metaphase.
t Standard error in parentheses.
marked decidual swelling. A notable feature was the presence of large invasive
trophoblastic giant cells.
Ten (28%6 %) out of 35 embryos sectioned, however, were in a state of advanced
degeneration. This proportion is not significantly different from 25 % (#2 =
0-24). In all cases apparently normal decidual responses had been induced,
although the embryos themselves had become little more than disorganized
masses of necrotic cells and cellular debris.
Of particular interest, however, was the presence of trophoblastic giant cells
embedded in the decidual tissue immediately around the embryonic remains
(Fig. 1 d). These giant cells were a feature of all the degenerate implantation
sites, and the majority appeared normal in respect of both size and morphology
when compared with those of litter mates.
The highly disorganized state of the great majority of cells in these embryos
precluded any meaningful analysis of cell number or mitotic index; however,
their comparatively advanced state of degeneration makes it seem unlikely that
any significant amount of growth or successful cell division had taken place
during the preceding 24 h.
Air-dried preparations
Forty three air-dried preparations of 4^-day blastocysts from Os/ + x Os/ +
matings were examined. In general, these consisted of large numbers of scattered
nuclei, interspersed by a few metaphase spreads.
Nine (21 %) of the preparations were very different in respect of two criteria.
Firstly, they displayed an extremely high proportion of metaphase-type spreads
sometimes in such abundance that it was almost impossible to distinguish one
set of chromosomes from another (Fig. 2a). Of those preparations sufficiently
Fig. 1. Embryos from Os/+ x Os/+ matings sectioned in utero (stained H & E).
(a) Day-5 Os/Os embryo displaying numerous mitotic-type cells. Attachment to the
uterus via the polar trophoblast is evident, (b) Day-5 Os/Os embryo which has
collapsed, (c) Normal litter mate of (a) displaying only one mitotic cell (M). (^Remnants of 6-day Os/Os embryo. Apparently normal trophoblastic giant cells (T) are
present in the surrounding decidual tissue.
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H. F. PATERSON
Studies on oligosyndactylism (Os) in mouse
121
well spread to be countable, a mean mitotic index of 34 % was calculated, in
contrast to the figure of 2 % obtained for their normal litter mates. (It should
be noted that mitotic indices may be prone to underestimation in these preparations, since vigorous cell spreading is apt to lead to the loss of integrity of
chromosome groups.) Secondly, within each of these embryos, the morphology
of the chromosomes differed widely, ranging through varying degrees of condensation from normal-looking metaphase spreads to tiny blobs of chromatin
scarcely larger than a centromere. Chromosomes were, however, of uniform
appearance within any single metaphase. The more normal types of spread
contained approximately 40 telocentric pairs of sister chromatids (mean = 38-6),
whereas the chromatin blobs of the very highly condensed types were nearly
always present in groups of about 80 (mean = 76-4). Close scrutiny of this
latter kind suggested strongly that they had arisen from the separation and
extreme spiralization of previously paired sister chromatids, since in some cases
the chromatin blobs were still loosely associated in pairs (Fig. 2b). This interpretation is also supported by the fact that chromosome number never exceeded
40 in the less condensed spreads containing clearly intact chromatid pairs.
As with the sectioned material, morphology of interphase nuclei in these
presumed mutant embryos appeared normal.
Culture over the lethal phase
(Data summarized in Tables 2 and 3)
The developmental behaviour of 149 zona-free blastocysts from Os/ + x Os/ +
matings, and of 96 from Os/+ x + / + matings was observed during culture
over the lethal phase.
No normal class was visible in either experimental or control groups at the
commencement of culture (4 days p.c). Typically, during the next 24 h, embryos
had become attached to the surface of the culture dish, and by 48 h (6 days^.c.)
were surrounded by a monolayer of outgrowing trophoblast. The inner cell
masses (ICMs) also became enlarged, and after 72 h of culture (7 days p.c.)
could be seen protruding upwards from the centre of the trophoblast layer.
In 36 embryos (24-2%) from inter-heterozygote matings, however, this
enlargement of the ICM failed to occur (Table 2). Indeed, after 48 h of culture
the ICM appeared markedly degenerate, and by 72 h had disintegrated almost
completely. In some cases clumps of necrotic cells remained clinging to the sur-
Fig. 2. Air dried preparations of Os/Os embryos (stained with orcein). (0) Spread
showing numerous mitotic cells, with chromosome morphology ranging from
normal metaphase type (M) to highly condensed type (C). (b) Highly condensed
group of chromosomes comprising 80 discrete blobs of chromatin. Signs of loose
association in pairs (P) support the conclusion that previously paired chromatids have
become become separated during condensation.
122
H. F. PATERSON
Table 2. Morphology of outgrowths after 72 h culture
Mating
OV + ?? xX Os/+ S6
o v + ?? +/+(?(J
Total no. of
Outgrowths
149
96
No. of out% total with
growths with
degenerate ICM degenerate ICM
36
2
24-2
2-1
Table 3. Measurements of outgrowths after 72 h culture
Class of embryo
Os/Os (II = 21)
Litter-mates (n = 70)
Controls (,n = 96)
Mean no. of
Trophoblast cells
Mean area of
Outgrowth *
Mean area/
Trophonlast cellf
14-2 ±1-4
27-2 ±1-2
91 ±1-2
16-3±0-7
17-6±0-5
0-65 ±006
0-59 ±002
0-64 ±002
28-7±0-8
* Area measured in arbitrary units corresponding to micrometer grid squares (1 unit =
4356 sq. /tm).
f Mean area/trophoblast cell = 2 (outgrowth area/trophoblast cell no.)/n.
(1) Mean trophoblast cell numbers of Os/Os and litter mates significantly different at
0-1% level (f89 = 5-4).
(2) Mean areas of outgrowth of Oa/Os and litter mates significantly different at 0-1 %
level (t89 = 4-9).
face of the trophoblast, but in others only a naked layer of trophoblast giant
cells remained. The morphology of these trophoblast cells seemed normal,
although there was a greater uniformity of size, arising from the absence of
smaller diploid or less giant, trophoblast cells which were present in the central
area of litter mates and controls. Viability too seemed unaffected since the cells
remained healthy and were of normal appearance after a further 24 h of culture.
By contrast, similar degeneration of the ICM was observed in only two of the
control embryos (2-1 %).
After proportionate correction for non-specific abnormality from the control
data, the x2 test indicates that the number of outgrowths with degenerate ICMs
in the experimental group (22-1%) is not significantly different from 2 5 %
(X2 = 0-66). It is thus concluded that these were Os homozygotes.
Since initial observations suggested that the trophoblastic outgrowth of these
mutant embryos was smaller than normal, direct measurements were made
both of the number of outgrowing trophoblast cells and of the area of outgrowth
after 72 h culture. This procedure also enabled a more accurate assessment to
be made of the average size of individual trophoblast cells in each group. A
total of 91 outgrowths from the experimental group (of which 21 were presumptive Os/Os), together with the 96 outgrowths from the control group were
thus analysed. The results show (Table 3) that the Os homozygotes are signi-
Studies on oligosyndactylism (Os) in mouse
123
ficantly smaller than litter mates and controls, both in terms of total size and
in number of trophoblast cells. There is, however, no evidence for a reduction
in mean trophoblast cell size in the mutant outgrowths.
DISCUSSION
The results of the histology in this study substantially corroborate those of the
original investigation performed by Van Valen (1966). In addition, the air-dried
preparations of Os/Os embryos confirm her suspicion that the abnormal cells
containing free chromatin bodies are blocked in mitosis; more precisely, they
appear unable to progress beyond metaphase.
Cell counts of day-5 embryos rule out any possibility that the high proportion
of metaphase cells seen in the mutants represents an increased rate of cell
division, since cell number is found to be substantially reduced. The morphology
of the chromosome spreads also opposes this; extreme chromosomal condensation as observed in Os/Os embryos is not seen in normally dividing cells but
may be produced by prolonged treatment with spindle poisons such as colcemid.
In particular it is significant that the discrepancy in mean cell number between
mutant embryos and litter mates (96 as opposed to 130) can almost exactly be
accounted for if the 40 % mitotic cells observed in the former group have been
blocked during division. These figures support Van Valen's conclusion that the
abnormality sets in during the seventh or eighth cleavage; for, if the mutation
affects all cells which have undergone a specific number of cycles, then allowing
for the accumulation of asynchrony during cleavage, a mean cell number of 96
indicates a blockage occurring at the eighth mitotic division.
A further significant feature of the histology was seen in uteri sectioned at
6 days. Whilst it is not surprising that the mutant embryos have induced a
decidual response, since some initial stages of implantation have taken place
on the previous day, the presence of apparently healthy trophoblastic giant
cells surrounding the implantation site of an otherwise necrotic embryo is of
considerable interest. The behaviour of Os/Os embryos in vitro mirrors the
events which take place in utero with respect to the persistence of trophoblastic
giant cells after the degeneration of the ICM. This refractoriness of giant cells
to the action of the lethal gene is most readily explained in terms of their cell
cycle, for these cells almost certainly attain their highly polyploid state by
endoreduplication (Chapman, Ansell & McLaren, 1972; Gearhart & Mintz,
1972). Trophoblast cells which have initiated endoreduplication prior to the
onset of Os gene action will consequently remain unaffected by a lesion which
exerts its principal influence on dividing cells.
The mitotic apparatus is a probable site of disruption in Os/Os cells for two
reasons. First, as seen in sectioned embryos, the chromosomes of affected cells
often exhibit a much more disorganized appearance than is observed in normal
metaphases, indicating that the spindle (if present) is incapable of organizing
124
H. F. PATERSON
the chromosomes into an orderly metaphase plate. Secondly, cultured wild-type
embryos, in which spindle formation is inhibited with colcemid, develop in a
very similar fashion to Os/Os embryos, exhibiting selective degeneration of the
ICM (Sherman & Atienza, 1975).
A singular aspect of the Os allele is that it interferes with a fundamental
function which has proceeded normally for several days prior to the lethal
phase. One explanation of such an effect occurring in early development is that
the mutant embryo's own genome may be unable to manufacture a substance
required for cell division, which has hitherto been present in sufficient reserves
within the cytoplasm. Such a substance would be subject, during successive
cleavages, to rapid and predictable dimunition in quantity per cell, thus accounting satisfactorily for the pronounced phase specificity exhibited by the Os
allele.
EM studies have demonstrated microtubular abnormalities associated both
with the somewhat analogous lethal-mitotique (Im) mutation in the newt, which
blocks cells in metaphase shortly after hatching (Gounon & Collenot, 1974),
and with lethal-polyploid (I(3)pl) in Drosophila hydei, which causes mitotic
disturbances in certain groups of cells during metamorphosis (Rungger-Brandle,
1911 a, b). A similar approach may prove able to reveal any structural peculiarities of the mitotic apparatus in Os homozygotes during the lethal-phase.
The author wishes to express his appreciation of the advice and encouragement given by
Dr Michael Snow and Dr Anne McLaren.
The work was supported by a Medical Research Council studentship.
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(Received 10 November 1978, revised 27 February 1979)
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