/. Embryol. exp. Morph. Vol. 32, 2, pp. 461-467, 1974
Printed in Great Britain
4ß1
Regional developmental capacities of the rat
embryonic endoderm at the head-fold stage
By A. S V A J G E R 1 AND B. LEVAK-SVAJGER 2
From the Institute of Biology, University of Zagreb
SUMMARY
Three areas, composed of all three germ layers, were isolated from Fischer strain rat
embryonic shields at the head-fold stage, and grafted separately under the kidney capsule
of adult male rats of the same strain. The areas were from the neural plate, Hensen's node
and the primitive streak. The resulting teratomas were examined histologically for the
presence of derivatives of the primitive gut.
The grafts differed strikingly in their capacity to develop into different segments of the gut.
Endoderm underlying the neural plate developed into derivatives of the foregut, while endoderm underlying the primitive streak developed mainly into derivatives of the mid- and
hindgut.
It was concluded that, at the head-fold stage, the capacities to develop into different
segments of the definitive gut are already roughly limited to particular areas of the endoderm.
INTRODUCTION
Exact knowledge of the organ-forming areas of the embryonic shield is
essential for any detailed experimental work on early embryos. This is particularly relevant to the endoderm because of the great variety of its numerous
derivatives.
It has been shown that in the early amphibian gastrula the endoderm already
shows the regionally specific capacity to differentiate into various sections of the
definitive gut (Holtfreter, 1938A, b). Using the method of grafting on the
chorioallantoic membrane, Rawles (1936) determined the organ-forming areas
in the chick blastoderm at the head-process stage. Through the'use of radioactively labelled grafts Rosenquist (1970, 1971a, b) succeeded in localizing the
prospective areas of different gut segments and their derivatives in the chick
epiblast at the primitive streak stage.
Mammalian embryos have not yet been subjected to this type of experiment.
Only recently a paper appeared dealing with the localization of the presumptive
gastric area in the endoderm of the rabbit embryo (David, 1971).
1
Author's address: Institute of Histology and Embryology, Faculty of Medicine, 41001
Zagreb, P.O. Box 166, Yugoslavia.
2
Author's address: Institute of Biology, Faculty of Medicine, 41001 Zagreb, P.O. Box 166,
Yugoslavia.
462
A. SVAJGER AND B. LEVAK-SVAJGER
Fig. J. A nearly sagittal section through a 9-day-old rat egg-cylinder. All., Allantois;
A, amnion; HN, Hensen's node; NP, neural plate; PS, primitive streak.
The present paper deals with the regionally restricted developmental capacities of the rat embryonic endoderm at the head-fold stage.
MATERIAL AND METHODS
The inbred Fischer strain of albino rats was used in the experiment. Eggcylinders belonging to the head-fold stage (stage 15 of Nicholas, 1962, fig. 1)
were isolated from uteri of females in the morning of the 10th day of pregnancy.
The ectoplacental cone and the Reichert's membrane were removed. The
embryonic shield was separated from the extra-embryonic part by an oblique
cut below the insertion of the amnion. Three areas, composed of all three germ
layers, were isolated from the embryonic shield, as demonstrated in Fig. 2.
These areas contained the neural plate (graft I), Hensen's node (graft II) and
the primitive streak (graft III) respectively. An intermediate region of the shield
was discarded. Each of the three areas was grafted separately under the kidney
capsule of a syngeneic adult male rat. After 15 days the recipient animals were
Regional capacities of rat
endoderm
463
Separate grafts
Fig. 2. Schema of the dissection of the 9-day rat egg-cylinder in order to isolate the
three areas of the embryonic shield. The orientation of the egg-cylinder as in Fig. 1.
killed and the teratomas were dissected from the host kidney. They were fixed
in Zenker's fluid, embedded in paraffin wax, serially sectioned, stained with
haemalum and eosin, and examined for the presence of differentiated tissues,
with special emphasis on the derivatives of the primitive gut.
RESULTS
The general characteristics of resulting teratomas were the same as of those
originating from grafted whole embryonic shields (Skreb, Svajger & LevakSvajger, 1971) or separated germ layers (Levak-Svajger & Svajger, 1974). The
teratomas derived from grafted Hensen's node area were remarkably smaller
than those derived from the other two areas. The results of the histological
differentiation within the teratomas are summarized in Table 1 and partly
illustrated in Figs. 3-6.
In Table 1 it can be seen that the three isolated areas of the embryonic
shield do not differ essentially in their capacity to differentiate into ectodermal
and mesodermal tissues. The only differences were: the poor development of the
skin in the Hensen's node area grafts, of the brown adipose tissue grafts from the
area of the neural plate, and of the smooth muscle for both of these areas.
On the contrary, the particular isolated areas of the embryonic shield differ
strikingly in their capacity to give rise to various derivatives of the primitive gut.
These derivatives did not develop in grafts of isolated Hensen's node area. In
teratomas derived from the area of the neural plate, only derivatives of the
anterior part of the foregut have developed (Fig. 3). Respiratory tube (ciliated
columnar epithelium + cartilage + glands) was the most prominent structure in
these teratomas. On the contrary, the grafted area of the primitive streak gave
rise predominantly to derivatives of the mid- and hindgut. Tubes and/or cysts
lined with a histologically well-differentiated mucosa of the small and large
30
E M B 32
Figs. 3-6. Details of the histological structure of teratomas obtained from the areas of the neural plate (Fig. 3) and of the primitive streak
(Figs. 4-6). B, Bone; Br, brain; C, cartilage; Gl, glands; Int, intestine; M, skeletal muscle; Pr, epithelial buds of the prostate; RT,
respiratory tube; Sk, skin; St, stomach ; Thy, thymus; UGS, urogenital sinus.
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Regional capacities of rat endoderm
465
Table 1. Differentiation of mature tissues in homografts of isolated areas of the
rat embryonic shield at the head-fold stage
Graft series
A.
Area of the embryonic shield
...
No. of successful grafts
Ectodermal tissues
Skin
Neural tissue
Mesodermal tissues
White adipose tissue
Brown adipose tissue
Cartilage
Bone
Smooth muscle
Skeletal muscle
Endodermal tissues
Oral cavity,* pharynx, oesophagus
Respiratory tube
Glands
Thymus
Stomach
Intestine
Urogenital sinus
I
Neural
plate
II
Hensen's
node
III
Primitive
streak
10
12
11
10
10
2t
12
11
11
9
It
10
10
0
10
12
7
12
10
1
12
11
10
10
11
10
11
9
10
8
4
0
0
0
It
0
0
0
0
It
0
0
0
0
0
3
10
6
* Partly of ectodermal origin (tongue as endodermal derivative).
t Rudimentary.
% The only oesophagus in all three series.
intestine, and surrounded by a typical two-layered muscularis, were regularly
found in these teratomas (Fig. 4). Derivatives of the urogenital sinus: the
urinary bladder (or urethra) and epithelial cords resembling rudiments of the
male genital glands, differentiated in close association with the large intestine
(Fig. 5). The stomach was found in only 3 out of 11 grafts of this series (Fig. 6).
DISCUSSION
The advantages of this method of grafting early rat embryonic material
under the kidney capsule and the reliability of the obtained results have been
discussed elsewhere (Skreb et al. 1971; Levak-Svajger & Svajger, 1974).
The fact that each of the isolated and grafted areas of the embryonic shield
was composed of all three germ layers explains the presence of tissue derivatives
of all three germ layers in the resulting teratomas. The failure of Hensen's node
area to give rise to skin may be attributed to the fact that it included only a
small part of the ectoderm, which was already determined to form neural
30-2
466
A. SVAJGER AND B. LEVAK-SVAJGER
structures. The absence of smooth muscle in teratomas derived from both the
neural plate and Hensen's node areas is concomitant with the lack of the stomach
and the intestine in these teratomas. As yet we cannot explain the absence of
brown adipose tissue in teratomas derived from the area of the neural plate. It
is surprising that hardly any endodermal derivatives have developed in grafts of
Hensen's node area. This may result from the small number of endodermal cells
in this area, and their continuity with cells migrating through Hensen's node.
The clear-cut difference in the type of endodermal tissues present in teratomas
derived from different areas of the embryonic shield suggest that at the head-fold
stage the capacities to develop into particular segments of the definitive gut are
already limited to particular areas of the endoderm. The endodermal area underlying the neural plate seems to be determined to form the anterior part of the
foregut and its derivatives, while the area underlying the primitive streak gives
rise to the mid- and hind-gut derivatives. The area of the endoderm below
Hensen's node and the anterior end of the primitive streak might be supposed to correspond to the posterior part of the foregut (oesophagus and
stomach).
From our previous experiments (Levak-Svajger & Svajger, 1971, 1974) it
seems very probable that at the pre-primitive-streak and the primitive-streak
stages all presumptive endodermal cells are localized within the primitive
ectoderm. At the head-fold stage, however, the capacity to differentiate into
derivatives of the primitive gut is restricted to the endoderm. At this developmental stage, the anterior part of the endoderm (underlying the neural plate)
isolated and grafted together with the adjacent mesoderm (but without the
overlying ectoderm), gave rise only to the derivatives of the foregut (LevakSvajger & Svajger, 1974). This is consistent with the results of the present
experiment, and, moreover, rules out the influence of the ectoderm on the
differentiation of the primitive gut at this stage.
On the other hand, the presence of the mesoderm seems to be essential for
the differentiation of the endoderm (Levak-Svajger et al 1969; Levak-Svajger &
Svajger, 1974). It is therefore only reasonable to speak of the regionally specific
interactions between endoderm and mesoderm rather than of the regional
developmental capacities of the endoderm alone. The same statement seems to
be valid for the amphibian embryo (Nieuwkoop, 1973).
It must be mentioned that in the present experiment the ectoderm also displayed some regionally specific histogenetic capacities. Some ectodermal
structures belonging to the head (choroid plexus, vibrissae, tooth germs)
developed only in grafts of the anterior area of the embryonic shield.
Regional capacities of rat endoderm
467
ZUSAMMENFASSUNG
Regionale
des embryonalen
Endoderms
Entwicklungspotenzen
Ratte auf dem Stadium des
Kopffortsatzes
der
Aus dem Rattenembryonalschild auf dem Stadium des Kopffortsatzes wurden drei, alle
drei Keimblätter enthaltenden Regionen - die Region der Neuralplatte, des Hensenschen
Knotensund des Primitivstreifens- isoliert und unter die Nierenkapsel syngenetischer erwachsener Tiere verpflanzt. Die resultierenden Teratome wurden mit besonderer Berücksichtigung
der Derivate des Primitivdarms histologisch untersucht.
Die Transplantate einzelner Regionen unterschieden sich wesentlich je nach ihrer Potenz
sich in die verschiedenen Segmente des Primitivdarms zu entwickeln. Das der Neuralplatte
unterliegende Endoderm hat sich in die Derivate des Vorderdarms differenziert. Das dem
Primitivstreifen unterliegende Endoderm differenzierte sich grösstenteils in die Derivate des
Mittel- und Enddarms.
Diese Befunde führten zum Schluss, dass die regionale Determinierung verschiedener
Segmente des Darms schon auf dem Stadium des Kopffortsatzes besteht.
We acknowledge the skilful technical assistance of Miss Durdica Cesar. This investigation
was supported by the grant IV/3 from the Research Foundation of S.R. Croatia, and in
part by N I H PL 480 Agreement No. 02-038-1.
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{Received 19 March 1974)