/ . Embryol. exp. Morph. Vol. 50, pp. 21-30, 1979
Printed in Great Britain © Company of Biologists Limited 1979
21
Effect of the T-mutation on histogenesis of the
mouse embryo under the testis capsule
By H. FUJIMOTO 1 AND K. O. YANAGISAWA1
From the Laboratory of Cell Biology, Mitsubishi-Kasei
Institute of Life Sciences, Tokyo
SUMMARY
Mouse embryos homozygous for the T-mutation show abnormalities, severer at the
posterior embryonic regions, by day 9 of gestation and die before day 11 in utero.
To analyse developmental potentiality of the T/T embryos, fragments of their anterior
and posterior portions were grafted into the testes of adult T/ + mice, and examined histologically for the tissues formed after 1 month.
The grafted tissues of the T/T embryos grew beyond the destined lethal stage and gave
rise to benign teratomas composed of mature tissues. Although there were some different
features of the tissues formed in the teratomas derived from different portions and stages of
the embryos, their types were essentially identical between wild-type and the mutant teratomas. Statistical analysis showed that frequency of the cartilage and/or bone formation
was significantly lower in the posterior mutant teratomas. It cannot be concluded, however,
that this difference is essentially caused by T-mutation. The main conclusion of present
experiments is that grafted portions of T/T embryos have the potentiality to develop intoteratomas containing derivatives of all three germ layers.
INTRODUCTION
In day 8-9 of gestation, mutants homozygous for the !T-gene are distinguishable from normal litter-mates by morphological abnormalities, which show a
cephalo-caudal gradient, in the axial organ system. The posterior end is more
affected than is the anterior (Chesley, 1935). Spiegelman (1976) has revealed
with the electronmicroscope that by day 9, chordamesodermal cells of the T/T
embryo form extensive tubular epithelial structures resembling the neural tube
in the posterior region, normally occupied by the notochord and the somites.
She speculated that these morphological abnormalities of the T/T embryo
might be caused by the incomplete specification of the primitive streak,
derivatives.
The T/T embryos die by day 11 because of the failure to establish placenta!
circulation as a result of abnormalities of the allantois (GluecksohnSchoenheimer, 1944). Therefore, it is impossible to observe histogenesis of the
T/T embryos in utero. If they were able to survive beyond the lethal stage,.
1
Authors' address: Laboratory of Cell Biology, Mitsubishi-Kasei Institute of Life Sciences,.
11 Minamiooya, Machida-shi, Tokyo 194, Japan.
22
H. FUJIMOTO AND K. O. YANAGISAWA
could their tissues showing morphological abnormalities differentiate into any
normal adult tissues ?
One method of investigating differentiation in early postimplantation embryos
of the rodents is by transferring them to extra-uterine sites, e.g. into the anterior
chamber of the eye (Grobstein, 1951), under the testis capsule (Diwan &
Stevens, 1976) or under the kidney capsule (Levak-Svajger & Svajger, 1974;
Svajger & Levak-Svajger, 1974). In these sites, the grafts survived long enough to
observe their final histogenesis. Present experiments were designed to analyse the
effect of the /"-mutation on histogenesis by transferring tissue fragments of the
T/T embryos to adult testes.
MATERIALS AND METHODS
The Brachyury mutant mice were originally supplied by Dr D. Bennett.
Adult mice for the present experiments (T/+ and + / + ) were obtained by
heterozygous matings (77+ xT/ + ) .
Pregnant female mice were sacrificed on days 8 and 9 of gestation (day of
vaginal plug = day zero). Embryos were removed from their decidua, and their
yolk sac and amnion were removed in Hanks' balanced salt solution under a
dissecting microscope. The T/T embryos obtained by heterozygous matings
(T/+ xT/+) were distinguished from their normal litter-mates by their typical
abnormal morphology. Their development was staged by the number of somites
of their normal litter-mates. The embryos from homozygous matings ( + / +
x + / + ) served as controls.
For morphological observation of the embryos, the removed embryos were
usually fixed in Bouin's fluid and embedded in paraplast. They were serially
sectioned at 7 /on and stained with haematoxylin and eosin. To observe finer
details of younger embryonic tissues, the embryos were fixed in 2-5 % glutaraldehyde followed by 1 % osmium tetroxide and embedded in Epon (Luft, 1961).
Sections, 1 /im thick, were stained with Ito's solution (Ito & Winchester, 1963).
To investigate histogenesis of the embryonic tissues, anterior and posterior
portions were dissected from + / + or T/T embryos using fine needles in
Eagle's minimum essential medium and grafted into the testes of adult T/+
male mice according to Stevens' method of surgery (Stevens, 1968). One graft
was dissected from each embryo. The recipient animals were killed one month
after grafting. Visible growths were dissected out, while testes without visible
growths were removed intact. They were fixed in Bouin's fluid and embedded in
paraplast. Sections, 10 pan. thick, were stained with Alcian blue at pH 2-5,
haematoxylin and eosin. For the examination of nervous tissue, the silver
impregnation method was carried out (Otsuka, 1962). All sections were examined under a light microscope for the presence of differentiated tissues.
Tissue types were compared with those described in a textbook of histology
(Bloom & Fawcett, 1975), and identified according to the descriptions of
Stevens & Hummel (1957).
Effect ofT-mutation on histogenesis
23
Fig. 1. Sagittal 1 /tm sections of the primitive streak region of the T/T'embryo at the
5 to 6-somite stage, x 160. Inset shows the rosette-forming cells, x 620.
RESULTS
In order to identify suitable stages for grafting into testes, development of the
T/T embryos was morphologically examined with special reference to the
unusual tubular structures at the posterior regions.
At the early somite stage, the'primitive streak area of the T/T embryo was
conspicuously enlarged. From histological observation, it was assumed that
mesodermal cells at the caudal extremity of the primitive streak area were
arrested (as found by Spiegelman (1976)). At the stage when normal Utter-mates
have five to six somites, small rosette structures consisting of several cells are
found in the posterior region of the T/T embryo (Fig. 1). At more advanced
stages, they develop into tubular structures (Figs 2 A, B). By about the 24somite stage, most chordamesodermal cells arrange themselves into tubular
structures, but somites are not formed. Necrosis is severer at the posterior end
of the embryo. Although the T/T embryo grows until about the 30-somite
stage, the portion posterior to the forelimb buds degenerates. Finally the T/T
embryo dies at about the 36-somite stage.
On the basis of such observation, grafting experiments of the T/T and wildtype embryos were undertaken at the following two stages. One was the, 4- to
10-somite stage (day 8) when the unusual tubular structures began to be formed.
The other was the 15- to 21-somite stage (day 9) at which this structure had
almost been established, yet necrosis in the posterior region of the T/T embryo
had not become too marked.
The anterior and posterior portions of the embryos at these stages were
dissected as shown in Fig. 3, and grafted into the adult testes. All successful
grafts resulted in tumorous growths. Size of the growths from the posterior
embryonic portions was generally larger than that from the anterior in each
genotype. Size of the growths derived from the T/T embryos was slightly
smaller than that from wild-type embryos at each stage.
24
H . FUJIMOTO AND K. O. YANAGISAWA
Fig. 2. Seven /*m sections of the posterior region of the T/Tembryo at the 14-somite
stage. The chordamesodermal cells form tubular epithelial structure. (A) Sagittal
section. The abnormal tube connects with the neural plate at its front region, x 110.
(B) Cross-section. x410. CA, caudal artery; H, hind gut; NP, neural plate; T,
tubular epithelium.
Histological observation showed that these growths were benign teratomas
composed of various mature tissues, but not embryonal carcinoma cells. The
features of histological organization were essentially identical in both genotypes.
There were, however, differences between the features of tissues formed in the
teratomas derived from the anterior embryonic regions and those from the
posterior.
Skin associated with hair follicles and sebaceous glands was conspicuous in
the posterior teratomas (Fig. 4A). It occupied a small area in the anterior
teratomas. Melanocytes were present in the dermis and the basal layer of the
epidermis. Melanocytes in the posterior teratomas were more abundant than in
the anterior. Adult nervous tissues were main components of the anterior
teratomas at day 9 (Fig. 4B). They occupied peripheral area of the teratomas
from other stages or portions of embryos. In some cases, neurons appeared to
have formed end plates on striated muscle (Fig. 4C). Ganglionic cells were
found among developing tissues (Fig. 4D). The number of ganglionic cells in
anterior teratomas was less than that of the posterior ones. Adipose tissue and
Effect ofT-mutation on histogenesis
25
D
Fig. 3. Diagram to illustrate dissected regions of the embryos. They were cut with
fine needles along broken lines. Arrows indicate anterior and posterior grafted
portions of the embryos. (A) Wild-type embryo at day 8. (B) The T/T embryo at
day 8. (C) Wild-type embryo at day 9. (D) The T/T embryo at day 9.
connective tissue were observed surrounding each organoid. Striated muscle
cells, cartilage and bone with marrow are found in the adipose tissue (Fig. 4E).
They were prominent structures in the anterior teratomas. Blood cells in capillaries were found in many sections, though it was not possible to determine
whether these tissues belonged to the teratoma or to the host. Smooth muscle
layers are observed encircling cavities lined with epithelium (Fig. 4F). These
epithelia in the posterior teratomas contained mainly goblet cells secreting
mucus, while they were mainly keratinized in the anterior. Usually, the epithelia
of these two types were adjacent to each other. They were referred to as alimentary tubes. Respiratory tubes, comprising ciliated columnar epithelium with
glands encircled with cartilage, were characteristic structures in the anterior
teratomas (Figs 4G, H).
From these histological observations, types of tissues identified in each
teratomas are scored in Table 1. The grafts of both genotypes gave rise to
mature tissues derived from all three germ layers. In many cases, single teratomas contained all the tissues listed in Table 1, but in some teratomas certain
of the tissues were missing. At day 9, 55 % of the posterior mutant teratomas
26
H. FUJIMOTO AND K. O. YANAGISAWA
Effect ofT-mutation on histogenesis
27
Table 1. Incidence of mature tissues found in teratomas
Age of the embryo
...
Day 8
Day 9
A
A
Grafted portion of the embryo
...
Posterior
Anterior
Anterior
Genotype of the embryo ...
No. of successful grafts
Ectodermal derivatives
Epidermis
Hair follicles
Sebaceous glands
Nervous tissue
Ganglionic cells
Melanocytes in dermis
Mesodermal derivatives
Cartilage and/or bone
Striated muscle
Smooth muscle
Adipose tissue
Connective tissue
Endodermal derivatives
Epithelium of alimentary tube
Epithelium of respiratory tube
T/T
Posterior
A
A
•>
r
+ / +• T/T
+ / + T/T
19
19
21
21
18
19
19
19
19
16a
17
19
19
19
19
12a
17
21
21
21
2P
21
21
21
21
19
18b
21
20
10e
8
9f
18
4
12*
19
19
18
19
18
19
19
17
19
19
20 c
21 d
21
21
21
14c
20
21
21
18
18
1
18
16
16
6
18
8
20
0
18
0
0
17
1 7
d
+ / +• T/T
18
20
20
5e
4
20
20
20
20
19
20
20
20
20
20
19
20
18
18
0
18
18
20h
20
19
20
20
10h
19
20
20
20
0
18
18
0
20
0
4f
18
6
98
a,x" = l-22,P>0-2;b, x 2 = 1-44,P>0-2; c, x = 3-86, P < 0 0 5 ; d, x2 = 2-49, P > 0 0 5 ;
e, x2 = 183, P > 0 1 ; f, x* = 1*93, P>0-l; g, x2 = 0-46, P>0-3; h, x2 = 108, P<0-01.
X" test; after Yates (1943) for small numbers.
FIGURE 4
Tissues in teratomas produced by fragments of the TIT embryos in the testis.
(A) Skin from a posterior portion of the embryo at day 9. Keratinized epithelium
with hair follicles, sebaceous glands, dermal connective tissue and adipose tissue.
x90.
(B) Nervous tissue from an anterior portion of the embryo at day 9. Stained with
silver impregnation method, x 180.
(C) Nerve and striated muscle from an anterior portion of the embryo at day 9.
Stained with silver impregnation method, x 360.
(D) A ganglion embedded in adipose tissue from a posterior portion of the embryo
at day 9. x 360.
(E) Endochondral ossification and red bone marrow in a teratoma derived from an
anterior portion of the embryo at day 8. x 72.
(F) Intestine from a posterior portion of the embryo at day 9. Epithelium contains
goblet cells, x 360.
(G) Respiratory tube from an anterior portion of the embryo at day 9. x 57.
(H) Ciliated columnar epithelium. Enlargement of a part of (G). x 360.
A, adipose tissue; B, bone; C, cartilage; CO, connective tissue; DC, degenerating
cartilage; G, gland; N, nerve; S, seminiferous tubule of the host; SM, smooth
muscle; ST, striated muscle.
28
H. FUJIMOTO AND K. O. YANAGISAWA
(11/20) were defective in some of the tissues examined, compared with 15 % in
the wild-type (3/20). In the mutant teratomas, missing tissue was cartilage
and/or bone in 99 % of the cases (10/11). The frequency of cartilage and/or
bone formation is significantly lower (P< 001, Table 1) in the mutant compared
to wild-type. Similarly, differentiation of these tissues is lower in the posterior
mutant teratomas at day 8 (i><005, Table 1). Of the posterior teratomas 67 %
(14/21) were deficient in some tissues examined, compared with 10 % in the
wild-type (2/21). In the mutant teratomas, 5 out of 14 defective teratomas
failed to form only cartilage and/or bone. Two were deficient in cartilage
and/or bone and other tissues, and seven were deficient in other tissues.
Accordingly, the posterior mutant teratomas lacking cartilage and/or bone
differentiation are not always deficient in other components. Defective tissues
varied in the mutant teratomas at day 8 compared to those at day 9. In contrast,
no significant difference was observed in the tissues examined between wild-type
and the mutant teratomas from the anterior embryonic portions (P>005,
Table 1). Although formation of ectodermal derivatives except nervous tissues
was remarkably reduced in the anterior teratomas of both genotypes at day 9,
the reasons for this reduction are not clear.
DISCUSSION
In the present experiments it was clearly shown that tissue fragments of the
T/T embryos developed into benign teratomas containing mature tissue derivatives from three germ layers. Frequency of differentiation of cartilage and/or
bone was significantly lower in the posterior mutant teratomas, compared to
the wild-type, while these tissues differentiated well in the anterior mutant
teratomas.
Bennett (1958) showed that the somites of the T/T embryo were defective in
the capacity for cartilage induction by reconstituting dissociated somites and
neural tube. She also reported that the forelimb buds of the T/T embryos
formed well differentiated cartilage. Thus there is a possibility, in our experiments, that the cartilage and/or bone formed in the mutant teratomas is derived
from the lateral mesoderm and that the somitic mesoderm has no ability to
form these tissues. It is difficult, however, to decide whether the cartilage and/or
bone in the teratomas are derived from the somites or the lateral mesoderm.
We cannot tell, therefore, whether this defect is a genuine effect of T-mutation.
Lethality of the T/t locus mutants has been a primary concern of many
investigators. Ephrussi (1935) cultured the T/T embryos in organ culture
beyond the stage of lethality in utero. Yanagisawa & Fujimoto (1977) isolated
cell lines from the T/T embryos. These results are all in accord with our present
observations.
Artzt & Bennett (1972) grafted t™18/^18 egg cylinders, in which no healthy
mesoderm was observed, into the testes of adults. The teratomas formed contained no mesodermal derivatives. In vitro culture of blastocysts derived from
Effect ofT-mutation on histogenesis
vl8
29
w18
matings producing r
homozygotes suggested that /
homozygotes are
viable beyond the normal period of lethality of the mutants in utero (Wudl,
Sherman & Hillman, 1977). On the other hand, t12, t* or ?w5 embryos were
lethal in both in vitro culture and grafting in ectopic sites (Wudl & Sherman,
1976; Wudl et al. 1977). Thus it is likely that the late-acting mutations (fwl8, T)
behave in a different way from the early-acting mutations (t12, te, tw5). We
cannot, however, exclude the possibility that some fraction of embryonic cells
of the T/T mutants is lethal and that the other fraction of cells survives beyond
the destined lethal stage.
A summary of this work was presented in a preliminary form at the 8th International
Congress of the International Society of Developmental Biologists in Tokyo, Japan, in
August 1977. We acknowledge with thanks Drs T. Miyake and T. Noguchi for their helpful
advice and constant encouragement. We are also indebted to Miss C. Murosaka for looking
after the mutant stock and technical assistance of histology.
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EMB
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2
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{Received 23 March 1978, revised 30 October 1978)