/ . Embryo!, exp. Morph. Vol. 47, pp. 39-52, 1978
Printed in Great Britain © Company of Biologists Limited 1978
39
Gastrulation in the mouse: assessment of cell
populations in the epiblast of twl8/tw18 embryos
By M. H. L. SNOW1 AND D. BENNETT 2
From the MRC Mammalian Development Unit, University College
London and Sloan Kettering Institute for Cancer Research,
New York
SUMMARY
W18
Homozygous / embryos die prior to organogenesis. They develop gross abnormalities
shortly after primitive streak formation. Anatomically, the lesion appears to be confined to
the mesoderm with that tissue showing ultrastructural deficiencies and abnormal migration
(Spiegelman & Bennett, 1974), and failing to develop in teratomas produced from mutant
embryos (Artzt & Bennett, 1972). Analysis of growth rate by determining cell number
increase, and by mapping mitotic activity and planes of cleavage in the epiblast shows that
the mutant embryos are small but paradoxically show overall a very high mitotic activity,
approximately double that of their normal litter mates. They also show a marked disorientation of the planes of cleavage in most of the epiblast.
In pre-primitive streak embryos, before gross abnormality is detectable, two types of
embryo can be found. One group constitutes the small embryos which also show the mitotic
disturbances characteristic of the later stage mutants. The second group, larger embryos, do
not show mitotic abnormalities. The rw|8 allele thus seems to act several hours before primitive streak formation.
Since there is no difference in the amount of cell death between mutant and normal
embryos until 6-75 days p.c. it seems that arrest in division is the cause of the elevated
mitotic index in mutants.
Significantly a small region of the epiblast in mutant embryos is free of the mitotic
abnormalities characteristic of the tissue as a whole. This region is the so-called proliferative
zone (Snow, 1977) and the data suggest that it may be from this region that some of the
ectoderm of the later embryos is produced.
The appearance of the primitive streak at the onset of gastrulation in the
mouse heralds a 24 h period of extremely rapid growth (Snow, 1976). During
that time, from 6-5 to 7-5 days post coitum, at least in the random bred Q strain
of mice, the epiblast, an apparently uniform population of about 600 cells,
generates the ectoderm and mesoderm of the pre-somite embryo which total
about 15000 cells. The definitive endoderm, which in the foetus replaces the
primary endoderm (Gardner & Papaioannou, 1975), has not yet been identified
at this stage.
1
Author's address: MRC Mammalian Development Unit, Wolfson House, University
College London, 4 Stephenson Way, London NW1 2HE, U.K.
2
Author's address: Sloan-Kettering Institute for Cancer Research, Laboratory of Developmental Genetics, 1275 York Avenue, New York, New York 10021, U.S.A.
40
M. H. L. SNOW AND D. BENNETT
The rate of cell division within the embryo shows regional variation and a
small area in the 6-5-day epiblast, constituting about 9 % of the whole tissue,
may generate about half the cells in the 7-5-day embryo (Snow, 1977). Cell
generation times of 2-3 h are estimated for this so-called proliferative zone,
which is located in the mid-line slightly towards the anterior end of the embryo.
The developmental fate of cells originating at this site is not known.
Of the several lethal mutations at the T-locus in the mouse (see Bennett, 1975)
the recessive /9 group, typified by the allele ?wl8, offers a possible means of
analysing more fully the cell populations involved in gastrulation. The homozygous /w18 embryo dies shortly after the primitive streak stage, apparently
because of abnormalities in mesoderm production (Bennett & Dunn, 1960;
Moser &Gluecksohn-Waelsch, 1967; Spiegelman & Bennett, 1974, and Spiegelman, 1976). The ectoderm seems largely unaffected by the mutation since if
embryos of this complementation group are transplanted to ectopic sites
tumours develop which are usually exclusively of ectodermal tissues only
occasionally containing any traces of mesoderm or endoderm (Artzt & Bennett,
1972).
MATERIALS AND METHODS
Heterozygous mice ( + /twls) were mated inter se and pregnant females
killed on the 7th and 8th day of pregnancy. Mating and fertilization are assumed
to occur between midnight and 0200 h and the approximate age of the embryos
estimated accordingly. After dissection from the uterus, the decidua containing
the embryos were fixed in Bouins fluid and processed for wax embedding.
10 /im thick sections were cut transversely to the embryo and stained with
haematoxylin and eosin. The analyses of cell numbers and mitotic activity
(metaphase/anaphase Index) were carried out as previously described (Snow,
1976, 1977), and the procedures are outlined in Figs 1 and 2. Briefly, serial
transverse sections were drawn using a camera lucida and all metaphases
and anaphases marked, showing the plane of cleavage (see Fig. 2d). From these
drawings tissue volumes were estimated using a planimeter and cell volumes
determined from high power drawings. Cell numbers are computed from these
data (see Fig. 1). The maps of mitotic activity were constructed as shown in
Fig. 2. Each embryo is regarded as being a stack of eight slices (histological
sections and divisions are assigned proportionately to the slices), which is then
cut down its lateral sides (Fig. 2b) and opened out flat. The luminal surface of
the epiblast/ectoderm would then appear as in (Fig. 2c). It is known how many
cells and divisions occur in each compartment so M/A indices can be calculated.
Some of the material analysed was that of Bennett & Dunn (1960) on which
the initial observations of the /w18 allele were made.
Gastrulation in t wl8 /t w18 mouse
41
embryos
Cell number
Fig. 1. Schematic representation of the method used for estimating cell numbers in
the various regions of the embryo. Each transverse section is drawn and the areas of
the tissue measured with a planimeter and volumes calculated according to section
thickness.
RESULTS
Seven entire litters ranging from 6-25 days to 7 days p.c. were processed; a
total of 59 embryos. Complete, undamaged serial sections were obtained from
50 embryos. In addition five appropriately sectioned and stained 7-5-day
embryos (two normal, three mutant) from three litters used by Bennett & Dunn
(1960) were analysed. The 6-25-day embryos are pre-primitive streak stages and
therefore could not be analysed with respect to the distribution of mitotic
activity. All 6-5-day embryos were very early primitive streak stages.
Cell number and mitotic activity
In the litters of 6-75 days and older the mutant and normal embryos can be
distinguished on morphological criteria and so comparisons of cell numbers and
mitotic activity were made. These data are shown in Table 1. For the 6-75-day
and 7-day-old groups, the data refer only to the epiblast and ectoderm cell
numbers in the embryo and do not include any of the mesodermal derivatives.
By 7-5 days the mutant abnormalities obliterate the boundary between ectoderm and mesoderm in many parts of the embryo so cell numbers refer to all
derivatives of the epiblast, i.e. ectoderm plus mesoderm. In the 6-75-day group
the Student /-test shows the difference in cell number and mitotic activity
between mutant and normal embryos to be highly significant (tls = 3-16,
42
M. H. L. S N O W A N D D. B E N N E T T
.1".
^
h.r.
h.r.
•p.s.
c
Fig. 2. Diagram illustrating the construction of the maps used in plotting the distribution of mitotic activity (A-C), and a representative transverse section (D) showing
the three modal directions of cleavage. The division at the top of (D) tends to
increase the length of the epiblast, the one at the right tends to increase the girth, and
the one at the bottom would lead to a thickening of the tissue (see text), p.s. =
primitive streak, h.f. = presumptive head-fold.
Table 1. Cell numbers and mitotic activity in morphologically identifiable
t wl8 /t w18 embryos (t/t) and their normal ( + /?) litter mates
i6-75
Age (days)
Type
No. embryos
Mean cell no.*
(±ls.e.)
Mean M/A Index
(±ls.e.)
70
+ /?
t/t
15
5
1350
2035
(115)
5-3
(0-2)
038)
7-8
(0-4)
70
+ /?
t/t
+ /?
t/t
5
1
1939
2
11764
(1611)
41
(0-6)
3
2487
(61)
5-7
(0-4)
6-8
5163
(859)
7-5
(1-2)
* In 6-75- and 7-day embryos this figure refers only to the epiblast, but in 7-5-day embryos
it refers to ectoderm plus mesoderm (see text).
Gastrulation in t wl8 /t w18 mouse embryos
43
Table 2. Frequency and distribution of cell divisions acting to thicken the epiblast
(see text and Fig. 2) in normal and mutant 6-75- and 7-day embryos. The data are
pooled from three litters.
Values with different superscript letters are significantly different (P < 001) by
the Student Mest.
'Thickening' divisions (:%)
Normal
r
18
No.
embryos
No. divs/
embryos
Primitive
streak
Lateral
Headfold*
20
6
116 ±8
103 ±10
39-3a
541 C
22-8b
35-P
18-6"
43-7°
* Presumptive head-fold region.
P < 001 for cell number and ?18 = 5-46, P < 0001 for M/A Index). At 7 and
7-5 days the differences are significant for cell number (P < 002) but not for
mitotic activity, although all the mutant M/A indices are considerably higher
than any normal M/A index.
In addition to this paradoxical combination of small size and high mitotic
activity, mutant embryos are abnormal with respect to the orientation of
dividing cells. In normal embryos cleavage planes which would act to thicken
the epiblast (Fig. 2d) comprise about 25% of the divisions and they are nonrandomly distributed in primitive streak stages with the primitive streak quarter
showing significantly more of such divisions (P < 0-01). In mutant embryos the
frequency of these divisions is higher and the distribution much more uniform
(Table 2).
These data are summarized as a histogram in Fig. 3. The litters are
numbered for identification and the embryos lettered according to cell number,
(a) being the smallest. All the mutants (encircled) show extreme values within
their litters for all three parameters. 6-25-day and 6-5-day embryos are morphologically homogenous but when histograms are drawn as for Fig. 3 then two
classes of embryo can be distinguished (Fig. 4). The single 6-5-day litter is
composed of two types of embryo based simply on the orientation of divisions,
(see also Table 3). A similar exercise indicates that eight of the 6-25-day embryos
are very small and six of these also show extreme values for at least one other
parameter.
The embryos showing characteristics of later stage mutants have been designated Class 2 and the others, generally with properties very different to the
mutants, Class 1. Overall these two Classes differ significantly from one another
for all parameters at 6-5 days and for the paradoxical low cell number and high
mitotic activity as 6-25 days (Tables 3 and 4). The data for 6-25-day embryos in
Table 4 are presumptive and potentially misleading. It is known that the longitudinal axis of the embryos will form transversely to the uterus but it is not
44
M. H. L. SNOW AND D. BENNETT
l_
%T
-
% T divs.
3g
3j
3i r 2b> 3h
^ >
3c lc 2g
S
2h 1b lh
2i
0
0
2e
2d
3f
3e
(2b)
2g
21"
li
lj
3d
3c 2h
If
1b
Ig
lh
3b
Id
Ik
le
4}
2e
2C
( )
(2;v
1
If
1
3e
2h
2g
le
Ig
3d
If
li
2f
Id
Ib
2e
3b
0
0
2d
U ^"YT^Y^I^I
1
3c
3d
Id
2f
If
lh
2h
3f
(1.)
2e
le
Ig
li
lj
1
<4
4
.5
:I
2i
3c)
2cJ
Id)
1
M A index
3c
tI
Fig. 3
If
lh
3h
le
2g
2c
lc
Ig
Id
2f
0
_ _©
:
1
1
l
3g
3i
C3 N)
3h
2g
3j
2f
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lh
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2e
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Ig
2h
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3c
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Ik
lc
1
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Cc II no.
(2a
2i
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2h
Qb)
—0
40
2f Mi?)
1
3e
3b
1
i
Ce Ino.
2d
lb
(2a
lc
2d
lh
3f
i2a
(/
Ig
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M 'A index
2i
Ik
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(3b)
t
(F)
3i
3c
2i
^0
3c
2d
divs.
<4
I
4\
:
1
ii
:)
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I
4 >4
Fig" 4
Fig. 3. The distribution of 6-75- (litters 1 and 2) and 7-day (litter 3) embryos with
respect to their litter means for cell number, M/A Index and frequency of 'thickening' cleavage divisions. Embryos are lettered according to cell number in each litter,
(a) being the smallest. The circles represent morphologically identifiable mutants.
The arrow indicates the litter mean and each class interval is 1 standard error.
Fig. 4. As Fig. 3, for 6-25-day (litters 1 and 2) and 6-5-day (litter 3) embryos. The
circles represent the identified Class 2 embryos (see text).
Gastrulation in t wl8 /t w18 mouse
45
embryos
Table 3. Analysis of embryos prior to the development of morphological deformity.
See text for definition of Classes 1 and 2
Superscript letters as for Table 2, P < 001.
6-25
6-5
Age (days)
Class 1
Class 2
Class 1
Class 2
No.
Mean cell no.
Mean M/A Index
'Thickening' divisions
(%)
11
513±21 a
51 ±0-3 a
23 ± 3 a
6
304±37 b
8-2±l-5 b
32±6 a
5
868 ± 46a
5-5±O-3a
17±2 a
5
669 ±73"
8-9±l-3 a
50±2 b
Table 4. Distribution of cell divisions acting to thicken the epiblast {see text and
Fig. 2) in the embryos featured in Table 3
Superscript letters as for Table 2, significance levels a-b, P < 005; a-c, P < 0001.
Region of embryo
Age (days)
6-25
6-5
Class 1
Class 2
Class 1
Class 2
Primitive
streak
Lateral
26-9a
37-5a
23-5a
50-4c
23-l a
28-2a
17-4a
52-7c
Headfold
17-7a
19.4a
11-4b
47.2c
possible to identify the polarity until the appearance of a primitive streak.
Therefore, in Table 4 it has been assumed that the quarter showing the higher
incidence of the 'thickening' cleavage is the future primitive streak quarter.
Although this is consistent with the evidence from later stages it is clearly an
artificial criterion. Even so, for these embryos the' primitive streak' and' headfold'
quarters do not differ significantly from one another with respect to the cleavage
planes of their dividing cells.
The difference between the two classes of embryo is enhanced when the
distribution of mitotic activity within the epiblast is considered. Figure 5 shows
distribution maps of M/A Index in normal and mutant embryos from the
6-75-day- and 7-day-old litters. The fw18 homozygote embryos show an abnormal
distribution of mitotic activity and in them it is not possible to distinguish the
proliferative zone seen in normal embryos. Similar differences are found at
7-5 days and also between classes 1 and 2 in the 6-5-day-old litter. The distribution maps for the 6-5-day embryos are shown in Fig. 6, and the M/A indices
along the mid-line have been plotted graphically in Fig. 7.
EMB 47
46
M. H. L. SNOW AND D. BENNETT
3-6
4-5
3-5
6-5
7-6
8-2
30
6 1
4-4
6-3
8-6
7-3
5-3
6-3
4-2
60
9-6
8-8
3-6
60
5-6
4-8
9-4
7-8
4-6
6-5
4-2
7-2
9-3
5-3
5-3-
71
61
5-8
10-3
5-5
41
8-7
6-7
7-6
7-4
8-7
5-5
91
7-5
6-5
10 6
11-6
7-9
81
5-6
6-3
6-2
9-6
7-2
5-6
4-4
5-9
4-7
10 6
8-2
6-9
5-6
60
5-7
8-6
10 8
7-9
5-5
50.
5-5
10-4
8-5
8-2
5-6
5-4
4-2
80
6-3
6-4
54
41
5-2
10-2
101
9-4
3-5
6-7
4-4
7-7
7-7
7-7
3-6
6-2
4-6
71
7-7
60
Fig. 5. Distribution of M/A Index (%) in the epiblast of 6-75/7-day embryos. (A)
normal, (B) /W18 homozygotes.
Table 5. Frequency of cell divisions acting to thicken the epiblast (see text and
Fig. 2) in the proliferative zone of normal and mutant embryos
Superscript letters as for Table 2.
Age (days)
6-5
6-75
and 7
Class 1
Class 2
Normal
/w18
No.
embryos
'Thickening'
cleavages (%)
5
5
20
6
32-6±6-3a
32-7 ±7-5 1 1
22-6 ± 2-1 *
27-8±7-7a
Cell death
Excessive cell death could account for the small size of mutant embryos so
an attempt has been made to quantitate this factor. Figure 8 shows crosssections through a normal and a homozygous fwl8 embryo both showing
evidence of cell death. Dead cells in the amniotic cavity can be easily scored but
cell boundaries are indistinct in the epiblast. For this tissue therefore the
Gastrulation in twl8/tw18 mouse embryos
47
00
7-1
4-9
1-9
12-2
90
07
40
7-6
71
12 6
110
1-6
8-3
10-2
5-3
11-7
110
2'2
21 1
6-5
4-4
22-1
9-3
5-5
14-3
3-8
2-4
14-3
8-9
3-7
10 8
4-7
9-3
6-7
80
7-2
9-7
2-1
8-0
9-6
12 6
5-5
4-5
11-9
4-2
8-3
6-3
11-6
5-9
3-2
3-2
8-9
40
8-8
5-5
3-2
7-8 •
6-3
8-3
13 9
10 8
3-9
6-7
2-2
6-5
12-4
4-9
7-6
9-6
2-8
9-4
13-4
4-6
3-6
3-8
5-5
50
13 7
4-8
0-3
4-1
00
8-7
12-4
0-8
10-8
7-3
2-8
00
8-1
30
1-3
6-6
3-6
00
140
9-2
Fig. 6. A s Fig. 5 for Class 1 (A) a n d Class 2 (B) e m b r y o s in the 6-5-day litter.
numbers of pycnotic granules have been counted. These data are shown in
Table 6.
DISCUSSION
The analysis of morphologically identifiable fw18 homozygotes indicates that,
in addition to the mesodermal abnormalities described by Bennett & Dunn
(1960) and Spiegelman & Bennett (1974), they contain significantly fewer cells
than their normal litter mates and show significant disturbances in the mitotic
characteristics of the epiblast. In litters taken prior to the development of
morphological deformity it is possible to distinguish two types of embryos;
a group with lower than average cell nembers but high mitotic activity like the
mutant embryos of later stages and a group with above average cell numbers
which do not show such mitotic disturbances. It seems reasonable to regard the
former group as the mutant twls homozygotes and the latter as the normal
embryos.
If this conclusion is valid then the time of expression of the fwl8 allele is
significantly earlier than previously believed and antecedes the first appearances
of mesoderm cells by at least 6 h. Consequently, the first signs of abnormal
development are observed in the epiblast. In order to assess whether the mitotic
4-2
48
M. H. L. SNOW AND D. BENNETT
i
Posterior
i
i
i
i
i
i
I
I
i
I
l
Anterior
Fig. 7. Comparison of mid-line mitotic activity in normal or Class 1 (O
O)
embryos and their /wl8/w18 or Class 2 (
) litter mates. The vertical bars represent ± 1 standard error.
disturbances are sufficient to account for the poor growth and death of the
mutant embryos it is necessary to consider mechanisms for their production,
and what consequences these might have.
Dealing first of all with the orientation of cleavage planes. The epiblast is a
single cell thick pseudo-stratified epithelium so at first sight the cleavage plane
tending to thicken the tissue seems anomalous. If the direction of division is
random and reorientation of cells takes place after cleavage then one-third of
divisions would be expected in this category. In the normal embryos there are
significantly fewer than one-third of such divisions, implying some underlying
control. Further evidence for such control is seen in Tables 2 and 4 which show
Gastrulation in twl8/tw18 mouse embryos
49
Table 6. Frequency of dead cells in normal and mutant embryos
Superscript letters as for Table 2, P < 0 0 1 .
No. of dead cells*
<
No.
6-25
Jitter 1
litter 2
Class
Class
Class
Class
1
2
1
2
6
3
5
3
Epiblast
57 ±9
44 ± 15
97 ± 13
45 ±8
Amniotic
cavity
Total
4±10
4+1-7
2 ±0-7
11 + 7 0
61
48
99
56
10-2a
14-3a
16-9a
3-1"
4.4a
6-5
litter 1
Class 1
Class 2
5
5
20±6
19±5
8 ±3-0
12 ± 4 0
28
31
6-75
litter 1
Normal
9
2
6
3
55 ±10
100 ±24
136± 14
156 ±52
6± 1-2
20 ± 5 0
1 ± 0-8
11 ± 4 0
61
120
137
167
litter 2
Normal
Epiblast
(%)
14.9a
3-2a
9.6b
5-6a
9.9b
* A 1:1 relationship between pycnotic granules and dead cells has been assumed.
Fig. 8. Transverse sections of a normal (A), and mutant (B) 6-75-day embryo. Dead
cells are alearly visible in the amniotic cavity and pycnotic granules (arrows) can
be seen in the epiblast of both.
50
M. H. L. SNOW AND D. BENNETT
a significantly higher proportion of these divisions found in the primitive
streak. In this region the logical outcome of such a plane of cleavage does indeed
seem to occur, i.e. the tissue thickens and cells are pushed through the primitive
streak. In contrast to the normal embryos the mutants have significantly more
than one-third of their divisions in this plane. Consequently it cannot be argued
that there is a simple loss of control over the division process in twl8 homozygotes but rather that there is a positive drive to orientate cleavage in this
direction. If an increase in the proportion of cells dividing in this direction is
a prerequisite for primitive streak formation then it could be expected that r wl8
homozygotes may form multiple or diffuse primitive streaks. Such a development could account for the extensive loss of definition between epiblast and
mesoderm which is a characteristic of the mutant embryo. In normal embryos
this loss of distinction between the tissues is confined to the primitive streak.
The elevated M/A index of the homozygous ?wl8 embryos could be accounted
for in several ways. (1) The cell proliferation rate may increase, (2) cell division
could be synchronous or (3) a greater proportion of the cell cycle may be
occupied by metaphase and/or anaphase. Of these possibilities the third would
appear to be the mechanism involved. An increase in cell proliferation rate
would result in larger embryos, not smaller. This paradox could have been
resolved by excessive cell death but Table 5 shows clearly that the paucity of
cells in mutant 6-25- and 6-5-day-old embryos cannot be ascribed to this factor.
Although there are approximately twice as many dead cells in the older fwl8
homozygotes this is thought to be a secondary effect, the mutant allele having
been expressed for some time by this stage (see below).
Synchrony of division can be discounted as none of the mutant embryos
displayed a low M/A index. It is extremely unlikely that these embryos, from
ten different litters were all caught in the division stages of their cycles.
It seems reasonable, therefore, to conclude that many mutant cells are slow
completing division and may not survive the process. The increase in dead cells
seen in post-primitive streak embryos is believed to be a consequence of this,
with cells persisting for a time in metaphase or anaphase and dying if division is
not completed. A similar lesion is observed in Os/Os embryos whose development is arrested about 4-5 days p.c. (Van Valen, 1966; H. Paterson, personal
communication). Clearly not all cells block and die in division because the
fwl8 homozygotes do grow, albeit poorly, between 6-25 to 7-5 days. Is this
growth from unaffected cells or does it simply represent the numbers that
struggle through this period? From Figures 4-6 it can be seen that the M/A
index is not elevated over the entire epiblast but that the region showing highest
mitotic activity in the normal embryos (the so-called proliferative zone (Snow,
1977)) does not show an increase. If the raised M/A Index had been caused by
accelerated proliferation rate then this could have been simply explained by
postulating that there is a maximum division rate, the result of physiological
constraints unconnected with the mutations, which has already been achieved
Gastrulation in twl8/t*18 mouse embryos
51
in the PZ. Since the elevated M/A index appears to be the result of blockage in
division alternative explanations must be sought. Only two seem possible.
Either the cells in the PZ are already blocked in division or these cells are unaffected by the lesion operative in the remaining epiblast. The kinetics of cell
production in the embryo between 6-5 and 7-5 days do not permit retardation
of proliferation in the PZ (Snow, 1977) so the second alternative pertains. It is
also noteworthy that the planes of cleavage appear undisturbed in the PZ of
twlB homozygotes (Table 5). Previous studies (Snow, 1977) suggest that normal
growth of the PZ region alone would be sufficient to account for the increase
in cell numbers observed in mutant embryos. It is difficult to resist drawing
attention to the fact that it is the ectodermal component of the ?wl8 homozygote that seems normal in teratomas (Artzt & Bennett, 1972).
Bennett, Boyse and Old (1972) suggested the involvement of T-locus genes
in sequential steps of development and Bennett (1975) has proposed that they
function by specifying cell surface components that are essential for the cellular
interactions in normal embryogenesis. Clearly, the changes in orientation of
cell divisions in the ?wl8 homozygote can most easily be explained in terms of
differential cell adhesions and are in accord with the hypothesis. However, it
is felt that the fundamental lesion in the Vs"18 mutant is the block to mitosis and
this is difficult to relate to cell surface phenomena. It seems more logical to
interpret this observation in terms of intracellular abnormalities in e.g. microtubules, spindle proteins or microfilaments. Spiegelman & Bennett (1974) have
identified microfilament abnormalities in t™18/^8 embryos and Dooher &
Bennett (1974), microtubular abnormalities in the semilethal r v2 homozygotes.
On the other hand the propensity of cells to divide may be coupled to topographical information they receive through their surface (e.g. primordial germ
cells in Sl/Sl homozygotes fail to migrate normally, apparently because of
defects in their environment; they also fail to proliferate at a normal rate
(Bennett, 1956)).
This work was carried out at the Sloan-Kettering Institute and I (MHLS) would like to
thank Dorothea Bennett for her generous hospitality and for making her laboratory facilities
so freely available. 1 am also grateful to DB and her colleagues, in particular to Karen Artzt,
Martha Spiegelman and Gerald Dooher, for much informative discussion about the T-locus.
The work was financed by a grant to DB from NSF.
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(Received 2 December 1977, revised 26 April 1978)