/. Embryol. exp. Morph. Vol. 40, pp. 271-276, 1977
Printed in Great Britain © Company of Biologists Limited 1977
271
Viability and metabolic activity of
homozygous Brachyury (T) embryos
By K. O. YANAGISAWA 1 AND H. FUJIMOTO
From the Laboratory of Cell Biology,
Mitsubishi-Kasei Institute of Life Sciences, Tokyo
SUMMARY
Some features of metabolic activity of homozygous mutants for the Brachyury (T) mutation
were studied. The embryos incorporated tritium-labelled thymidine up to about the '32-36
somite' stage. The total amount of protein per embryo increased until the same stage. T/T
cells proved viable in vitro over the in utero lethal period. Several cell lines were established
from + / + and T/T embryonic cells.
INTRODUCTION
The semidominant mutation T (Brachyury) at the T/t locus of the mouse
leads to a short-tailed phenotype when heterozygous and is lethal in homozygous condition. The homozygous embryos (T/T) go through the early stages
of embryonic development without showing any obvious abnormalities. By 9
days of gestation, TIT embryos are markedly abnormal. Their body ends just
posterior to the anterior limb-buds, the somites are diffuse, and the neural
tube is severely kinked (Chesley, 1935). To date this mutant has been studied
mainly from the morphological point of view and little is known about its
metabolic state (Griineberg, 1958; Bennett, 1964; Spiegelman, 1976). In order
to study these embryos at the cellular level (Yanagisawa & Fujimoto, 1977),
it was first necessary to know something of their metabolic state.
It is believed that the homozygous mutant embryos are lethal because they
fail to establish placental connexions (Gluecksohn-Schoenheimer, 1944).
Ephrussi (1935) showed that these embryos could grow in organ culture beyond
the time at which they would have died in utero. Gluecksohn-Schoenheimer
(1944) reported a similar result by explanting the embryos in the chick host.
The present investigation revealed that dissociated TjT cells are also viable in
vitro and cell lines have been established from both + / + and T\T embryonic
cell cultures.
1
Author's address: Laboratory of Cell Biology, Mitsubishi-Kasei Institute of Life Sciences,
11 Minamiooya, Machida-shi, Tokyo 194, Japan.
272
K. O. YANAGISAWA AND H. FUJIMOTO
MATERIALS AND METHODS
Mice and embryos. Origin and maintenance are as described by Yanagisawa
& Fujimoto (1977).
Analysis of mitotic figures. Essentially the same method was employed as
was described by Yanagisawa & Kitamura (1975).
Cell culture. Cells or tissue fragments were cultured in Eagle's minimum
essential medium supplemented with 10% heat-inactivated horse serum and
3 % chick embryo extract (MEMHC), in an atmosphere of 95 % air and 5 %
CO2.
Radioautography. Pregnant females were injected intraperitoneally with
7/tCi/g body weight of [3H]thymidine (specific activity 12-4 Ci/m mole) and
killed 1 h later. Embryos were removed quickly, washed thoroughly with
Hanks' salt solution and fixed in Bouin's fixative. They were embedded in
paraplast and serially sectioned at 5 /«n. Slides were washed with 5 % trichloroacetic acid, dipped in Sakura emulsion NR-M2 (Konishiroku Photo Ind. Co.,
Tokyo), and stored in a dark box at 4 °C for 6 weeks. They were developed
and stained with haematoxylin.
Determination of amount of protein. Embryos were collected in 0-3 ml of
0-002% Triton X-100 and frozen at - 7 0 °C. One tenth millilitre of 4 N NaOH was added to the thawed samples and the amount of protein was
determined by the method of Lowry, Rosebrough, Farr & Randall (1951).
RESULTS
Although TjT embryos look abnormal morphologically, they develop in a
regular fashion. Thus the size and shape of the head of T\T embryos change in
a similar way to their normal litter-mates and the forelimb-buds appear at a
similar stage (24-somite stage). Even degeneration of the posterior trunk is not
random, but starts when the normal litter-mates reach the 32-36-somite stage.
It was therefore possible to stage the mutant embryos, though less accurately
than the normals. Conventionally, stages of the mutant embryos were indicated
by the number of somites of their normal litter-mates.
The number of mitotic figures per unit area (10~2 mm2) was calculated for
embryos of 9 (15-26-somites), 10 (27-36-somites) and 11 days (37-40 somites)
of gestation. As shown in Fig. 1 for the level of the forelimb-bud, the number
of mitotic figures per unit area in TjT embryos is comparable to that of + / +
embryos up to 10 days (30-36-somite stage), but drops to zero at 11 days
(37-40-somite stage). The number of mitotic figures per 10~2 mm2 of the neural
tube at the posterior end of 10-day TIT embryos (average of 13 alternate sections from the tip) was 2-9 ± 0-4. Although this value is lower than that at the
level of the forelimb-bud, mitotic cells existed among necrotic cells at the
posterior end of the embryos.
Metabolic activity o/T/T embryos
273
150 -
100 •
Gestation period (days)
Fig. 1. Number of mitotic figures/10"2 mm2 in the neural tube of wild-type and
mutant embryos, at the level of the forelimb-bud. O, + / + ; • , TIT. Number of
embryos analysed are as follows: 9-day: +/ + , 5; TIT, 3; 10-day: +/ + , 2; TIT, 4;
11-day: +l + ,2;
T/T,3.
[3H]Thymidine was incorporated into the nucleus of T\T cells at 10 days
but no grains were found over the nucleus of 11-day T\T embryos. The percentage of labelled nuclei in the neural tube and the mesenchyme was counted
in 9-, 10- and 11-day embryos. Embryos of each gestational age were siblings.
The results are summarized in Table 1. In the homozygous mutant, around
60% of the cells were labelled. Even at the degenerating posterior end of T\T
embryos, over 50% of the cells were labelled in 9- or 10-day embryos. These
results coincide with those obtained by the analysis of mitotic figures, indicating
that the cells of the homozygous embryo have mitotic activity up to 10 days
of gestation.
18-2
274
K. O. YANAGISAWA AND H. FUJIMOTO
40 -
30
20
10
16
20
24
28
Number of somites
32
36
Fig. 2. Amount of total protein per embryo. O, + / + embryos; A, TIT embryos.
Numerals in the graph indicate the number of embryos used for determination of
each point. Standard error is indicated where more than duplicate determinations
were made.
Figure 2 shows the amount of total protein per embryo. TjT embryos have
less than their normal litter-mates, but the amount increases up to the 32-36somite stage.
Although the mutant embryos are resolved by 12 days of gestation, their cells
are viable if cultured in vitro. Dissociated T/T and + / + cells have already
Metabolic activity o/T/T embryos
275
Table 1. Percentage of labelled nuclei in the neural tube and
mesoderm of normal and mutant embryos exposed to [3H]thymidine
Day
10
Embryo
Level of section
% nuclei labelled
+/+
Forelimb-bud
77 +
TIT
Posterior end
Forelimb-bud
Forelimb-bud
+ 1+
Posterior end
Forelimb-bud
69-3 ± 2-8 (neural tube)
75-8 ±2-1 (mesoderm)
65-6 ±4-1 (neural tube)
71-2 ±2-8 (neural tube)
67-3 ± 4 1 (neural tube)
78-7 ±2-1 (mesoderm)
56-2 ± 4 1 (neural tube)
69-6 ±2-8 (neural tube)
79-2 ±1-9 (mesoderm)
64-2 ±4-1 (neural tube)
67-3 ±2-8 (neural tube)
59-2 ±5-2 (neural tube)
78-5 ±1-8 (mesoderm)
54-0 ±41 (neural tube)
79-2 ±2-1 (neural tube)
0 (neural tube and
mesoderm)
T/ +
TIT
11
+/+
T/T
Posterior end
Forelimb-bud
Forelimb-bud
Posterior end
Forelimb-bud
Forelimb-bud
been passaged over 50 times in 15 months. Preliminary results show that T\T
cell lines show some characteristics of freshly isolated TIT embryonic cells.
Cloning and further characterization of these lines are in progress.
DISCUSSION
Our results suggest that TIT embryos at the 12-36-somite stage are still
viable and that dissociated cells from these embryos can serve as experimental
material. Erickson & Pedersen (1975) have reported that tG/t* embryos can
survive beyond the in utero lethal period if they are cultured in vitro. On the
other hand, Wudl & Sherman (1976) have recently suggested that tw5ltw5 and
t12/t12 embryos are lethal at similar stages in vitro to in vivo. The present results
clearly demonstrate that at least some fraction of T\T cells are viable in vitro.
Further study of these lines may shed some light on the action of the T gene.
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K. O. YANAGISAWA AND H. FUJIMOTO
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ERICKSON,
(Received 5 January 1977, revised 16 March 1977)