/ . Embryol. exp. Morph. Vol. 42, pp. 127-134, 1977
Printed in Great Britain © Company of Biologists Limited 1977
127
Tissue differentiation
in ectopic grafts after cultivation of rat
embryonic shields in vitro
By N. SKREB AND V. CRNEK 1
From the Department of Biology, Faculty of Medicine,
University of Zagreb, Yugoslavia
SUMMARY
Differentiation of rat embryonic shields in vitro is inferior to that obtained in grafts under
the kidney capsule. To see whether the in vitro differentiation is stable or whether it can be
improved in a better environment, rat embryonic shields, isolated on gestation days 8 and 9,
were transplanted under the kidney capsule after a period of 7 or 14 days in vitro.
After transfer, some tissues appear in the kidney that never appear in vitro. Many but not
all tissues appear more frequently in grafts than in vitro. Most tissues differentiate further in
grafts than in vitro, though skeletal muscle, when cultivated for 14 days before transplantation,
is an exception.
Thus the effect on differentiation of imperfect culture conditions can be counteracted, for
some tissues but not all, by grafting to the kidney. The effect depends partly on the time spent
in vitro before transfer.
INTRODUCTION
One method of investigating differentiation in early postimplantation rodent
embryos is by transplanting them to extrauterine sites (Skreb, Damjanov &
Solter, 1972). The resulting teratoma-like structures provide evidence on the
conditions necessary for differentiation (Skreb & Svajger, 1975) and for the
formation of embryo-derived teratocarcinomas (Solter, Adams, Damjanov &
Koprowski, 1975).
Rat egg-cylinders transferred beneath the kidney capsule showed almost the
same capacity for differentiation whether they had two or three germ layers
(Skreb, Svajger & Levak-Svajger 1971), but transplanted into the anterior
chamber of the eye those with two germ layers showed quite different behaviour
from those with three (Levak-Svajger & Skreb, 1965). Although rat embryonic
shields cultivated in vitro for 2 weeks can give rise to many differentiated tissues
(Skreb & Svajger, 1973), the level of differentiation is inferior to that seen in
either the kidney or the anterior chamber of the eye.
1
Authors' address: Zavod za Biologiju, Medicinski Fakultet, 41000 Zagreb, Salata 3,
Yugoslavia.
9-2
128
N. SKREB AND V. CRNEK
The present experiments were designed to find out whether the inferior
differentiation of the postimplantation rat embryo in vitro can be improved by
subsequent transplantation under the kidney capsule.
MATERIAL AND METHODS
Fischer-inbred rats were used. Gestation was considered to have begun early
on the morning when sperm was found in the vaginal smear. The following
24 h was designated gestation day 1. Embryos were isolated from the uterus on
gestation days 8 or 9 when they are supposed to have respectively two or three
germ layers.
The extra-embryonic part and the ectoplacental cone were cut off at the level
of the amnion, and Reichert's membrane was torn. The embryonic shields were
put on a small piece of lens paper supported by a stainless-steel grid in an
embryological watch glass (Skreb & Svajger, 1973); this was placed in a simple
gas chamber (New, 1966). Eagle's minimum essential medium with 50% rat
serum was used as culture medium. Antibiotics were added in standard concentration, and the medium was changed daily. Explants were maintained
in vitro for 7 or 14 days. All 7-day explants were transferred under the kidney
capsule of a recipient adult rat about 3 months old (Skreb etah 1971). The 14-day
explants were divided into two series. One series of each developmental age was
fixed (Florez-Cossio, 1969), embedded in paraffin wax, serially sectioned and
stained with haemalum and eosin. The other series was transferred under the
kidney capsule. Fourteen days after transfer the animals were killed and the
grafted kidney removed. The resulting teratoma was isolated, fixed in Zenker's
fluid and processed as mentioned above.
We have used morphological criteria of differentiation only. Each cell or
tissue type was identified as specifically as possible, so that the incidence of
tissue types could be calculated. No attempt was made to estimate the amount of
the various tissues present in the explants of grafts.
We also tried to assess the degree of differentiation, in the sense used by
Wylie, Nakane & Pierce (1973). As terminal differentiation is a multiphasic
process, several definite steps can be recognized in some tissues (e.g. myoblast,
myotube, muscle fibre).
RESULTS
(1) 8-day rat embryonic shields {two germ layers)
(a) In vitro culture (14 days)
The explants consisted mainly of undifferentiated stroma with very few
identifiable tissues (Table 1, series 1). They were often covered either with
multilayered squamous epithelium which was not keratinized or with an endoderm-like layer of cuboidal and columnar cells (Fig. 1). Well differentiated gut
epithelium was never found, although some intermediary forms were sometimes
Series
No. of
cases
Days
in vitro Epidermis
Neural
tissue
Gut epithelium
Smooth
muscle
Skeletal
muscle
Cartilage
Bone
Adipose
tissue
8-day shields
Explants
—
—
1
35-7*
200
700*
—
4-2
—
70
14
3-7
90-5
71-7
37-7a
100
22-6
2
7
7-5
20-7
Grafts after dif53
870
5-5
5-5a
68-5
870
14-8
ferent culture
3
14
18-5
54
—
period
9-day shields
78-6
63-6
59-4c
19 6e
28-4
69-4
4
14
Explants
239
97-lb
971
800 d
971
100
82-8
5
7
971
35
971
Grafts after
75.7b. c
27-ld.e
100
96-9
96-9
57-5
6
14
78-7
different
33
63-6
culture period
* In series 1, stratified squamous epithelium was seen, rather than differentiated epidermis, and endoderm-like cuboidal cells rather than gut epithelium.
a 2
d 2
x = 16-41, P < 0001.
x = 1903, P < 0001.
b 2
e 2
x = 503, P < 005.
x = 0-613, P > 0-5.
c
x° = 3-26, P > 005.
Technique
Table 1. Tissues found in explants and ectopic grafts {expressed as the percentage of explants or grafts containing the tissue)
130
N. SKREB AND V. CRNEK
Tissue differentiation in rat in vitro
131
observed. Muscle was never present and cartilage only rarely. In almost 50 % of
explants haemopoiesis was present, and some trophoblastic cells were recognized. Such cells were never observed in 9-day rat embryonic shield cultures.
(b) 7 days in vitro and 14 days under the kidney capsule
The majority of transplants had many well differentiated tissues (Table 1,
series 2). Series 2 differs markedly from series 1, even with respect to the
epithelia. Gut epithelia show terminal differentiation (Fig. 3) and the epidermis
has a well keratinized layer. Smooth muscle and cartilage are often present,
though skeletal muscle is extremely rare. The incidence of cartilage is greatly
increased after transfer. Adipose tissue and bone were seen.
(c) 14 days in vitro and 14 days under the kidney capsule
In this series (Table 1, series 3), the degree of differentiation of epithelia
(epidermis and gut) is again higher than after cultivation in vitro only, and
resembles the differentiated epithelia in the preceding series. However, although
smooth and skeletal muscle and adipose tissue are as frequent as in the preceding series, the incidence of cartilage is very low, as low as after in vitro
culture only. Skeletal muscle is very rare and bone is not seen.
(2) 9-day rat embryonic shields {three germ layers)
Series 4 (see Table 1) gives the previously published results of cultivation
in vitro of 9-day rat embryonic shields (Skreb & Svajger 1973) for comparison
with the new series (e.g. Fig. 2).
The incidence of various tissues, particularly smooth muscle and adipose
tissue, is higher in series 5 and 6 of Table 1 than after cultivation only (series 4).
After cultivation only, skeletal muscle was represented by myotubes (Fig. 4),
FIGURES
1-6
Figs. 1-3. Various degrees of differentiation of gut epithelium.
Fig. 1. 8-day rat embryonic shield in vitro (14 days). Note the entoderm-like
appearance, 500 x.
Fig. 2. 9-day embryonic shield in vitro (14 days). Intermediary differentiation,
500 x.
Fig. 3. Kidney graft after 7 days in vitro (8- or 9-day embryo). Note the well differentiated intestinal epithelium, 210 x .
Figs. 4-6. Various degrees of differentiation of skeletal muscle (9-day rat embryonic
shield).
Fig. 4. In vitro (14 days). Note the myotubes in a connective tissue stroma, 500 x .
Fig. 5. Kidney graft after 14 days in vitro. Note the similarity to the myotubes in
Fig. 4, 500 x .
Fig. 6. Kidney graft after 7 days in vitro. Note the striated muscle fibres forming
bundles, 500 x .
132
N. SKREB AND V. CRNEK
but after culture for 7 days followed by transfer to the kidney capsule it was
well differentiated, with striated muscle fibres combining to form bundles
(Fig. 6). The incidence of skeletal muscle was also greatly increased (series 4
and 5, Table 1). On the other hand transfer under the kidney capsule did not
influence the incidence of skeletal muscle when the 9-day rat embryonic shields
had been cultivated for 14 days (series 6, Table 1), while terminal differentiation
was also less good than in grafts of embryonic shields that had spent only
7 days in vitro (Fig. 5).
DISCUSSION
In considering the embryo's capacity to differentiate outside the uterus
(Skreb & Svajger, 1975), it seems that both the incidence of various differentiated
tissues and the degree of differentiation is influenced by the developmental
stage of the transferred embryo and also by the type of extrauterine environment. The dependence on developmental stage is, in experiments presented
here, well illustrated by the behaviour of 8-day rat embryonic shield. In contradistinction to the 9-day shield it gives rise very rarely to bone or to skeletal
muscle in kidney grafts. The isolated rat embryonic shield interacts with different
environmental factors by undergoing 'more or less' differentiation, rather than
an 'all or none' reaction.
The second question is the stability of differentiation of isolated rat embryonic
shields. This has been analysed several times, but only reversibility in different
environments has been tested, leading to different and controversial conclusions
(Weiss, 1973; Wallace, Maden & Wallace, 1974; see Ursprung, 1968).
In contrast, we have been concerned not with the reversibility of differentiation, but with whether a weak phenotypic expression of differentiation can be
improved by a better environment. To our knowledge, the only similar experiments are those of Grobstein (1951). He explanted mouse embryonic shields
into Carrel flasks on a plasma clot consisting of chicken plasma, horse serum,
Tyrode's solution and chick embryo extract for 2-7 days, then implanted them
in the anterior chamber of the eye. As very few, if any, differentiated tissues
were found in his cultures, a comparison similar to ours was impossible. We
obtained different results for different tissues. Ectopic grafting of in vitro
explants generally improved the degree of differentiation of tissues already
present in the explants (e.g. squamous epithelium, gut epithelium). A conspicuous
exception is skeletal muscle when the rat embryonic shield had been cultivated
for 14 days before the transplantation. It resembled the myotubes of explants
more than terminally differentiated striated muscle fibres (Figs. 4 and 5).
As to the incidence of differentiated tissues after transfer under the kidney
capsule, some tissues appeared in the kidney that never appeared in vitro (e.g.
smooth, skeletal muscle, bone and adipose tissue in 8-day shields and bone
and adipose tissue in 9-day shields). The second group of tissues appeared more
frequently in grafts than in vitro, provided the culture period was restricted to
Tissue differentiation in rat in vitro
133
7 days. Besides several epithelia, this group is represented by smooth and
skeletal muscle and cartilage in 9-day shields and cartilage in 8-day shields.
The third group of tissues did not appear more frequently in the kidney than
in vitro. Besides neural tissue, which had about the same incidence in vitro and
in grafts, this group includes cartilage and bone in 8-day shields and cartilage
and skeletal muscle in 9-day shields, but only when cultivated for 14 days before
transfer. The results strongly suggest that the time spent in culture may have had
an effect on subsequent differentiation in the kidney grafts.
The incidence of some tissues increased only after 7 days in vitro, but not
after 14 days. Terminally differentiated tissues appear in the second week of
culture, heart and haemopoiesis excepted (Skreb, Scukanec-Spoljar & Crnek,
1976). After only 7 days in vitro very few cells may have undergone determination and hence a change of environment may have been able to change their
final differentiation. Cells that have been determined to follow one developmental pathway may simultaneously be restricted in their potential to express
other phenotypes (Wilkins, 1976). Thus the behaviour of skeletal muscle and
cartilage in 9-day shields and cartilage in 8-day shields may be explained by the
supposition that the determination of mesenchymal cells takes place during the
second week of cultivation, preventing any increase of incidence in ectopic
grafts after 14 days in vitro. This restriction is very stable, at least under our
experimental conditions. In this case we can speak about lost competence. We
do not know whether conditions can be found in which this competence can be
regained, or whether in vitro culture has produced an irreversible loss of
differentiative capacity.
On the other hand, the same incidence of differentiation in grafts is shown by,
for example, smooth muscle regardless of the time spent in vitro. Either the
cells may be already committed in vitro but show no phenotypic expression, at
least as judged by the morphological criteria used in this study, or some
mesenchymal cells may stay uncommitted in vitro, even for 14 days.
We thank Dr Anne McLaren for her helpful discussion and criticism.
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{Received 21 January 1977, revised 2 June 1977)