J. Embryol. exp. Morph. Vol. 66, pp. 43-55, 1981
Printed in Great Britain @ Company of Biologists Limited 1981
43
In vivo and in vitro development
of mouse embryos homozygous for the embryonic
lethal velvet coat (Ve) mutation
By J. ROSSANT 1 AND K. M. VIJH
From the Department of Biological Sciences, Brock University,
St. Catharines, Ontario
SUMMARY
Embryos homozygous for the velvet coat mutation, Ve/Ve, were recognized at 6-5 days
post coitum by the reduced size of the ectodermal portions of the egg cylinder and the loose,
columnar nature of the overlying endoderm. Later in development ectoderm tissues were
sometimes entirely absent. Abnormalities appeared in the ectoplacental cone at 8-5 days but
trophoblast giant cells and parietal endoderm appeared unaffected. Homozygotes could not
be unequivocally identified at 5-5 days nor at the blastocyst stage but were recognized in
blastocyst outgrowths by poor development of the inner cell mass derivatives, It has previously been suggested that Ve may exert its action at the blastocyst stage by reducing the
size of the inner cell mass, but no evidence for such a reduction was found. Most of the
observations on Ve/Ve homozygotes are, however, consistent with the hypothesis that Ve
exerts its action primarily on later primitive ectoderm development.
INTRODUCTION
Embryonic lethal mutations in the mouse have been widely studied in an
effort to gain insight into the genetic control of early developmental processes
(reviewed McLaren, 1976). One such early acting lethal which has not yet been
extensively studied is the semidominant mutation, velvet coat (Ve) (Stieler &
Hollander, 1972). In the heterozygote, Ve acts on the coat to produce a thick,
velvety appearance but Ve/Ve homozygotes die soon after implantation. Initial
reports suggested that Ve might act by preventing normal primitive ectoderm
formation. Diwan & Stevens reported in Mouse News Letter in 1974 and in
McLaren (1976) that Ve/Ve embryos could be recognized by the deficiency or
absence of primitive ectoderm cells at the time of implantation, although most
other tissues appeared normal. The embryos were resorbed by 9-10 days of
pregnancy without forming mesoderm. Diwan and Stevens also suggested that
Ve/Ve homozygotes might be recognizable at the blastocyst stage by their
reduced inner cell mass (ICM) size, although no data were presented to support
1
Author's address: Department of Biological Sciences, Brock University, St. Catharines,
Ontario L2S 3A1, Canada.
44
J. ROSSANT AND K. M. VIJH
this. This possible link between reduced ICM size and reduced or absent primitive ectoderm has made velvet coat an interesting mutation for study. Experimental studies on normal embryos have suggested that primitive ectoderm
versus primitive endoderm formation is determined by the position of
ICM cells relative to the blastocoel cavity (Gardner & Johnson, 1975;
Gardner & Papaioannou, 1975; Rossant, 1975). Cells that are on the outside of
the ICM (normally adjacent to the blastocoel) form primitive endoderm while
enclosed ICM cells form primitive ectoderm. If this positional interpretation is
correct, a reduction in ICM size should lead to a reduction in the proportion of
primitive ectoderm to primitive endoderm, since most cells would now be
'outside' cells (cf. ICM and trophectoderm formation at the blastocyst stage:
Tarkowski & Wroblewska, 1967). It is possible, therefore, that the major defect
in Ve/Ve embryos causes small ICM size and that the reduction in primitive
ectoderm stems directly from this. If Ve/Ve blastocysts can be recognized, this
hypothesis could be readily tested by injection of extra ICM cells, which should
allow rescue of the mutation.
Before such manipulative experiments can be undertaken, the reported effects
of the Ve mutation must be confirmed. Granholm, Stevens & Theiler (1980) have
recently published a description of the post-implantation development of the
velvet coat mutation on a C57B1/6J strain background which essentially confirms earlier reported findings. However, they did not look at pre-implantation
stages. In the present study, we provide a detailed description of the in vivo
and in vitro development of Ve/Ve embryos on a 129J strain background.
MATERIALS AND METHODS
Matings
The velvet coat (Ve) mutation (obtained from the Pasteur Institute, Paris) was
maintained on a 129J strain background by forced heterozygosity. Mutant
embryos were obtained by mating Ve/+ heterozygotes and control litters were
produced by reciprocal backcrosses of Ve/+ $x + / + $ and + / + ?x Ve/ +
$. No differences were found between embryos from the two backcrosses and so
all control results were pooled. Natural mating was used to obtain all postimplantation embryos but pre-implantation blastocysts were recovered after
superovulation to increase embryo numbers. Intraperitoneal injection of 5 i.u.
of pregnant mare's serum gonadotrophin (PMS, Organon) was followed 48 h
later by 5 i.u. human chorionic gonadotrophin (hCG, Sigma). Ovulation and
mating were presumed to occur approximately 12 h after hCG injection.
Examination of post-implantation embryos
Female mice from Ve/+ x Ve/+ matings were killed at 5-5, 6-5, 7-5, and 8-5
days post coitum and any implants werefixedin acetic formol alcohol, dehydrated
and embedded in wax before sectioning at 7 ptm. Sections were stained with
Development o/Ve mouse embryos
45
haemalum and eosin and examined with the light microscope. Female mice from
control matings were killed at the same stages of pregnancy but only 5-5-day
implants were processed for histology. Older control embryos were dissected out
intact and examined carefully under the dissecting microscope. This procedure
was adopted after dissection of 6-5 days and older embryos from Ve/+ x Ve/ +
crosses revealed that the major histological abnormalities detected in presumed
Ve/ Ve embryos could also be recognized in the intact embryo.
Examination, culture and transfer of blastocysts
Blastocysts were obtained from both Ve/+ x Ve/ + matings and control
matings 98 h after hCG injection and all were examined under the dissecting or
compound microscope for any obvious abnormalities. Some were classified
visually into early and late blastocysts and transferred separately to recipient
females on the 3rd day of pseudopregnancy. Recipients were killed on the 8th
day of pregnancy and examined for normal or abnormal fetuses. Some blastocysts were subjected to immunosurgery (Solter & Knowles, 1975) and the
resulting inner cell masses (ICMs) were fixed, air-dried (Tarkowski, 1966) and
stained with Giemsa for cell counts. Air-dried spreads of intact blastocysts were
also made. Other blastocysts from control and experimental matings were
cultured in tissue-culture dishes (Falcon) in a-modified MEM (Gibco) plus
10 % foetal calf serum, gassed with 5 % CO2 in air and maintained at 37 °C for 7
days. A further series of experimental and control blastocysts were cultured on
feeder layers of Mitomycin-C-treated STO fibroblast cells (Ware & Axelrad,
1972). After 7 days, most cultures were washed with PBS, fixed in acetic alcohol
and air-dried prior to staining with Methylene Blue. This enabled measurements of the diameters of the trophoblast giant cell nuclei to be made. A few
cultures were grown in solvent-resistant culture dishes (Permanox, Lux) and
then fixed in 2-5 % glutaraldehyde in 0-1 M phosphate buffer, post-fixed in OsO2,
dehydrated and embedded in Spurr's resin (Spurr, 1969). Sections 1 /im thick
were cut with a glass knife on a Huxley ultramicrotome and stained with a
mixture of toluidine blue and Azur II.
RESULTS
Post-implantation development
The post-implantation development of embryos from experimental and control matings is summarized in Table 1. Most control embryos were entirely
normal but a few resorbed or resorbing embryos were detected at each stage of
development. These generally consisted of clumps of dead cejls with a few
trophoblast giant cells. Similar early resorptions were observed in a few cases in
the experimental matings and these were not considered further. At 6-5, 7-5 and
8-5 days of development, histological examination of conceptuses from Ve/ +
x Ve/ + matings also revealed a class of embryos with distinct sets of abnorm-
46
J. ROSSANT AND K. M. VIJH
Table 1. Postimplantation development of embryos from Ve/+ x Ve/ +
and Vej + x + / + matings
Type of cross
Ve/+ x Ve/ +
Ve/+ x +/ +
Day of
development
examined
5-5
6-5
7-5
8-5
5-5
6-5
7-5
8-5
No.
No. of
embryos
22
41
49
28
35
41
43
37
obviously abnormal (%)
0
12 (29-2)
9(18-4)
8 (28-6)
0
0
0
0
No. resorbed
(%)
0
1 (2-4)
2 (41)
2 (71)
1 (2-8)
3 (7-3)
4 (9-3)
2 (5-4)
Fig. 1. (a) Section of 5-5-day embryo from Ve/+ x Ve/ + mating, classified as
'dumpy'. All tissues are present including giant cells and distal endoderm although
this is not obvious in this section. Uterine epithelium beneath embryo is highly
folded. (6) Section of 5-5-day control embryo from Ve/+ x + / + mating.
Development of Ve mouse embryos
47
50/im
Fig. 2. (a) Section of 6-5-day presumed Ve/ Ve embryo. Both embryonic and extraembryonic ectoderm appear reduced, leaving the proximal endoderm as a loose sac
at the bottom of the egg cylinder. All other tissues appear normal, (b) Section of control 6-5-day embryo.
alities not observed in control matings. This abnormal class constituted 24-5 %
(29/118) of all embryos examined at the three stages which is close to the
expected 25% Ve/Ve homozygotes and was therefore presumed to represent
these embryos.
At 5-5 days of development, no clear distinction could be drawn between
normal and mutant embryos. All embryos recovered were egg cylinders with
both extraembryonic and embryonic ectoderm present. The only embryos which
showed any sign of abnormality were four embryos which were classified as
'dumpy' (Fig. 1). These egg cylinders were smaller than their litter-mates and all
showed areas of infolded uterine epithelium beneath the embryo. However,
there was no obvious disproportionate growth of any one tissue and no sign of
excessive cell death. By 6-5 days of development presumed Ve/Ve embryos were
48
J. ROSSANT AND K. M. VIJH
^^^MSL
100/im
Fig. 3. Section of 7-5-day presumed Ve/Ve embryo. Ectoderm derivatives (e) are
reduced and misshapen and surrounded by loose, columnar proximal endoderm (p).
This section is not quite medial so that the ectoplacental cone is not clear but other
sections revealed that this tissue appeared healthy. Reichert's membrane and
adhering distal endoderm (d) are visible, surrounded by haemorrhagic tissue. The
parietal yolk-sac cavity appears full of debris.
Fig. 4. Section of 7-5-day presumed Ve/Ve embryo. This embryo contains no
obvious ectoderm derivatives and consists of a loose lump of proximal endoderm
(p) attached to the ectoplacental cone.
recognized quite readily by their shortened appearance and reduced embryonic
and extraembryonic ectoderm. The proximal endoderm overlying the embryonic
ectoderm was thickened and columnar instead of squamous. In some cases the
ectoderm tissues were so reduced that the proximal endoderm formed a loose sac
at the end of the egg cylinder (Fig. 2). In no case were embryonic or extraembryonic ectoderm completely absent and they always appeared healthy.
Mitotic figures were observed and no excessive cell death occurred. No other
tissues showed any obvious abnormalities.
Fig. 5. Section of 8-5-day presumed Ve/Ve embryo. Ectoderm is reduced, misshapen and surrounded by
columnar endoderm. No mesoderm is visible. Ectoderm-like structure (arrow) in ectoplacental cone.
Fig. 6. Section of 8-5-day presumed Ve/Ve embryo. Also misshapen and small but mesoderm (m) apparent
between ectoderm and endoderm. No obvious primitive streak. Again ectoderm-like structure (arrow) in
ectoplacental cone.
100 jum
I
-s,
b
50
J. ROSSANT AND K. M. VlJH
By 7-5 days, the mutant phenotype was more variable. Four of the nine
presumed Ve/Ve embryos were still egg cylinders but, as at 6-5 days, the ectoderm tissues were drastically reduced and the proximal endoderm was thickened,
loose and columnar. In one of the embryos the embryonic ectoderm was rather
misshapen (Fig. 3). The remaining five embryos showed little or no embryonic or
extraembryonic ectoderm. Other trophoblast tissues (ectoplacental cone and
giant cells) were visible, and so was distal endoderm. However, the embryonic
region consisted of a mass of loose, vesiculated cells which were presumed to be
collapsed proximal endoderm (Fig. 4). In two cases, there was perhaps a little
ectoderm inside the endoderm, but this was not very clear. A large amount of
haemorrhage was observed between the parietal endoderm and trophoblast.
At 8-5 days, two presumed Ve/Ve embryos were again represented by ectoplacental cone tissue and giant cells with small vesicles of proximal endoderm.
The remaining seven embryos had evidence of some ectoderm derivatives and
were presumably formed from Ve/Ve embryos that retained ectoderm at 7-5
days. In two cases the remaining ectoderm was a very small lump surrounded by
loose proximal endoderm (Fig. 5). This embryonic remnant was further surrounded by blood, debris and giant cells. Metaphases were observed in the
ectoderm lumps. The remaining four embryos contained more ectoderm although this was always very misshapen and surrounded by thick endoderm.
These four embryos all showed evidence of some mesoderm formation (Fig. 6).
No organized primitive streak or amniotic folds were observed, but cells of
mesoderm morphology were found as an intervening layer between endoderm
and presumed embryonic ectoderm. Five embryos (four with embryonic ectoderm, one without) also showed abnormal development of the ectoplacental
cone, which appeared to be organized into columnar epithelium-like structures,
resembling embryonic or extraembryonic ectoderm (Figs. 5, 6). No such
structures have ever been reported in normal ectoplacental cone development.
Blastocyst examination and transfer
Blastocysts from Ve/+ x Ve/+ matings were compared with blastocysts from
control matings and no consistent abnormalities were observed in the experimental group. Particular attention was paid to the size of the ICM but no
blastocysts with obviously reduced ICMs were observed, nor were there any
particularly small ICMs observed after immunosurgery. Cell counts, however,
revealed a slight difference between the mean cell number of control and
experimental ICMs (Ve/+ x + / + = 23-2±0-66, n = 79; Ve/+ x Ve/+ =
21-0 ±0-83, n = 86). Histograms of ICM cell number showed that 26-7% of
ICMs from Ve/+ x Ve/+ matings had fewer than 15 cells whereas only 11-3 %
of control ICMs fell into this class (Fig. 7). These two percentages were significantly different (x2 = 7-4, P < 0-01) but the difference between the two
values does not amount to the 25 % expected if all Ve/Ve embryos fell in this
cell number class. If the low ICM cell number apparently shown by some Ve/Ve
51
Development 0/Ve mouse embryos
510
IIIS
1620
2125
Cell no.
2630
3135
3640
8
s:
g:
3140
4150
o •
5160
o •
o
8o
o
6170
7180
8190
Cell no.
Fig. 7. Histograms of ICM and blastocyst cell numbers from Ve/+ x Ve/+ and
Ve/+ x + / + matings. (A) ICM cell numbers: (B) Blastocyst cell numbers.
• , Ve/+ x + / + : O, Ve/+ x Ve/ + .
ICMs were due to preferential reduction in ICM size, we would have expected to
recognize Ve/Ve ICMs by small size after immunosurgery which we failed to do.
We would also have predicted that intact blastocyst cell counts should not reveal much difference between control and experimental embryos, since three
quarters of the cells of the blastocyst are trophectoderm which would be unaffected. This prediction was also not fulfilled. There was a large difference in
mean cell number between control and experimental groups (Ve/+ x + / + =
66-5 ±2-6, n = 19; Ve/+ x Ve/+ = 54-0±2-9, n = 22). This difference
was due to the presence of seven embryos in the experimental cross with cell
numbers in the 30-50 range (Fig. 7) typical of early blastocysts (Rossant & Lis,
1979). Such retarded blastocysts would contain ICMs of normal size but low cell
52
J. ROSSANT AND K. M. VIJH
Table 2. Development of blastocysts from Ve/+ x Ve/+ and
Ve/+ x + / + matings after 7 days in vitro
Type of culture
a medium
a x STO cells
Type of cross
Ve/+ x Ve/ +
Ve/+x+/ +
Ve/+ x Ve/ +
Ve/+ x + / +
No. of
embryos
No. egg
cylinders
No. giant cells
± disperse cells
(%)
94
62
124
78
65
51
90
73
29 (30-8)
11(17-7)
34 (27-4)
5 (6-4)
number, explaining the results of ICM cell counts. There was, thus, no conclusive evidence for a specific reduction in ICM size in Ve/Ve homozygotes and
even general retardation of growth was not an infallible marker of Ve/Ve
embryos. Embryo transfers showed that 3 out of 8 retarded blastocysts from
Ve/+ x Ve/ + matings were Ve/Ve homozygotes while 4 out of 18 expanded
blastocysts also proved to be Ve/Ve.
In vitro culture
Although Ve/Ve homozygotes could not be unequivocally identified at the
blastocyst stage, they were recognizable after a few days in blastocyst outgrowths (Table 2). In a medium alone, 30-8% of Ve/+ x Ve/+ blastocysts
failed to show normal egg-cylinder development (ectoderm and endoderm
formation) and produced spreads of trophoblast giant cells with occasionally a
few disperse cells which were presumed to be ICM derivatives. It was thought
likely that the Ve/Ve embryos were included in this class but the rate of abnormal development in the control cultures was too high (17-7 %) to allow any
firm conclusions to be drawn. Improved development of control cultures was
achieved by using a feeder layer of fibroblasts; the percentage of control embryos showing poor ICM development was reduced to 6-4%. However, under
the same culture conditions, 27-4 % of Ve/ + x Ve/ + blastocysts failed to
develop normal ICM structures. If the control rate of abnormal development is
subtracted from this figure, 21% of the blastocysts from Ve/+ xVe/ +
matings show unexplained abnormal development. This is close to the expected
figure of 25 % Ve/Ve homozygotes. Trophoblast giant cell formation appeared
normal in all outgrowths from the experimental embryos. Comparison of the
nuclear diameters of all the peripheral trophoblast cells of each outgrowth
(Wudl & Sherman, 1978) revealed that the extent of endoreduplication was
similar in both normal and presumed mutant embryos (Table 3).
Development of Ve mouse embryos
53
Table 3. Nuclear diameters of trophoblast cells from presumed
mutant and wildtype blastocyst outgrowths
Type of outgrowth
Presumed Ve/Ve
Presumed Ve/+ or +/ +
No. of
outgrowths
measured
No. of cells
Mean nuclear
diameter
(/tm)±S.D.
5
5
49
62
52-4 ±13-5
52-8 ±12-9
DISCUSSION
The earliest stage at which embryos homozygous for the velvet coat mutation
could be unequivocally recognized in vivo in this study was 6-5 days post
coitum, when presumed Ve/Ve embryos showed reduced egg-cylinder growth.
Both embryonic and extraembryonic ectoderm were apparently reduced in size
and proximal endoderm overlying the embryonic region was loose and columnar
rather than squamous. We were unable to identify unequivocally Ve/Ve
embryos at 5-5 days of development as reported by other workers using mice of a
different genetic background (Granholm et al., 1980), although some embryos
appeared normal but retarded. We were also unable to identify Ve/Ve homozygotes prior to implantation. No class of blastocyst showing consistent abnormalities, including reduction in ICM size, was detected in Ve/+ x Ve/ +
matings. There were more normal but retarded blastocysts in experimental
matings than in control crosses, but embryo transfer studies showed that this was
not an absolute criterion for identifying Ve/Ve homozygotes. Thus, on our
strain background, the velvet coat mutation does not seem to exert any marked
effect on morphogenesis until well after implantation, although it may contribute to general retardation of growth at earlier stages. Our initial hypothesis
that the velvet coat mutation caused a reduction in ICM size and thence a
reduction in the ratio of primitive ectoderm to primitive endoderm does not
seem to be correct.
However, velvet coat might still provide some useful insights into normal
developmental processes if it could be shown to be tissue-specific, affecting only
the primitive ectoderm and its derivatives. Most early developmental lethals do
not appear to be restricted to one cell type or another (McLaren, 1976). All the
abnormalities observed in Ve/Ve homozygotes at 6-5 days could be interpreted
as secondary effects of a primary failure of primitive ectoderm growth, although
they are not confined to this tissue. Reduction in the size of the extraembryonic
ectoderm could be brought about by failure of the primitive ectoderm to provide the normal stimulus for proliferation (Gardner & Papaioannou, 1975;
Rossant & Lis, 1981) and the loose, columnar nature of the proximal endoderm
might be a response to reduced pressure from the underlying ectoderm. Later
54
J. ROSSANT AND K. M. VIJH
than 6-5 days, other abnormalities occurred that are harder to reconcile with
tissue specificity of Ve action, but their late appearance suggests that they too
could be due to secondary effects. In vitro studies also showed poor development of the primitive ectoderm from Ve/Ve blastocysts, although trophoblast
giant cell formation was not affected and outgrowths survived beyond the
normal time of death in vivo.
Although most of the description of Ve/Ve homozygotes is consistent with
the hypothesis that Ve acts specifically on the primitive ectoderm, morphology
alone cannot be used to establish the primary site of action of the gene. The
hypothesis must be tested experimentally by assessing the ability of various
tissues from Ve/Ve embryos to form chimeras with normal embryos. The
hypothesis predicts that both trophectoderm and primitive endoderm from Ve/
Ve embryos will be able to survive when combined with normal embryonic cells
but primitive ectoderm will not be able to contribute to a chimera. Our inability
to recognize unequivocally Ve/Ve homozygotes at the blastocyst stage and the
absence of any suitable genetic marker closely linked to Ve will make such
experimental analysis difficult (Papaioannou & Gardner, 1979).
We should like to thank Drs V. E. Papaioannou, L. C. Stevens, N. H. Granholm and
D. A. Hickey for useful comments on the manuscript. This work was supported by the
Canadian Natural Sciences and Engineering Research Council.
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(Received 16 June 1980, revised 8 April 1981)