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/. Embryol exp. Morph. Vol. 62, pp. 277-289, 1981
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Differentiation of the
yolk-sac endoderm under the influence
of the digestive-tract mesenchyme
ByTOHRU MASUI1
From the Zoological Institute, Faculty of Science,
University of Tokyo
SUMMARY
To reveal differentiation potency of yolk-sac endoderm, this tissue from quail embryos was
cultured alone or in association with digestive-tract mesenchymes of chick embryos.
When yolk-sac endoderm was cultured alone in vitro, the endoderm of the area vitellina
differentiated into the yolk-sac parenchyma, but the endoderm of the extraembryonic area
pellucida (EEAP) failed to differentiate into yolk-sac parenchyma, and the endoderm of the
area vasculosa became necrotic.
When endoderm of the area vitellina was cultured in association with digestive-tract
mesenchymes, all the endodermal cells developed into yolk-sac parenchymal cells after two
days. Later, basophilic cells appeared among them, and differentiated into both mesenchymespecific epithelia and intestinal-type epithelium with a striated border, and villi were also
formed. Goblet cells appeared in all types of recombinations. The endoderm of the EEAP
cultured with digestive-tract mesenchymes gave similar results to that of the area vitellina. In
contrast, endoderm of the area vasculosa, when cultured with digestive-tract mesenchymes,
became necrotic.
The present investigation demonstrated that the endoderms of the area vitellina and of the
EEAP differ in self-differentiation potency, and that their developmental fates can be modified by the influence of digestive-tract mesenchymes. These endoderms can differentiate into
the mesenchyme-specific epithelia, though they often differentiate also into the intestinal-type
epithelium.
INTRODUCTION
Epithelia of the extraembryonic membranes (such as amnion, chorion, and
allantois) of the avian embryo have been used to analyse the inductive ability of
various mesenchymes, and a vast number of dissociation and recombination
experiments have been carried out (Bonetti, 1959; Kato & Hayashi, 1963;
Kato, 1969; Mizuno, 1970, 1972; Sawyer, Abbott & Trelford, 1972; Mizuno &
Yasugi, 1973; Yasugi & Mizuno, 1974; Sawyer, 1975, 1978; Yasugi, 1976a, b,
1979; Dhouailly, 1978; Gumpel-Pinot, Yasugi & Mizuno, 1978; Fisher &
1
Author's address: Zoological Institute, Faculty of Science, University of Tokyo, Hongo,
Tokyo 113, Japan.
278
T. M A S U I
Yolk-sac areas
Embryo
Digestive organs
Embryo
GIZ
5-day chick embryo
3-day
quail embryo
5-aay
quail embryo
Endoderm
\
/
Selfdifferentiation
/
\
/
Control
Fig. 1. Diagram showing the mode of combination of yolk-sac endoderm with
various mesenchymes of the digestive tract. Abbreviations: OE, oesophagus; PRO,
proventriculus; GIZ, gizzard; SI, small intestine; EEAP, extraembryonic area
pellucida; AVas, area vasculosa; AVit, area vitellina.
Sawyer, 1979). However, the reactivity of epithelium of the yolk sac, in spite
of its importance in embryonic life, has received little attention.
It has been generally accepted that the yolk-sac endoderm differentiates from
the hypoblast and area opaca endoderm (Jacobson, 1938; Vakaet, 1970; Rosenquist, 1972; Wolk & Eyal-Giladi, 1977). In a previous investigation, we studied
the normal development of yolk-sac endoderm in the quail embryo and demonstrated that it develops from two different areas: the area opaca and the EEAP
(Masui, 1978).
In the present study, dissociation and recombination experiments were performed to reveal the differentiation potency of the endoderms of the area opaca
and of the EEAP, when they were cultured in vivo and in vitro in association
with or without various mesenchymes of the digestive tract.
MATERIALS AND METHODS
Embryos
Chick (Gallus gallus domesticus) and Japanese quail (Coturnix coturnix
japonica) embryos were used. Eggs were incubated at 37 °C. Yolk sac of quail
embryos and digestive organs of chick embryos were used throughout.
Differentiation of the yolk-sac endoderm
Oesophagus
Proventriculus
•
279
Small intestine
• Yolk-sac parenchymal cells
*^ -* Epithelium of mesenchyme-specific type
o—o Epithelium of small intestinal type
Fig. 2. Time course of differentiation of 3- and 5-day area vitellina endoderm cultured for 1 to 3 weeks in vitro in association with various mesenchymes of 5-day
digestive tract. The percentage of grafts showing mesenchyme-specific induction
and self-differentiation potency is compared to the total number of grafts recovered.
Isolation of tissue fragments
Endodermal fragments of the 3- and 5-day area vasculosa and of the 5-day
EEAP and mesenchymal fragments of the 5-day digestive tract were, isolated
completely with collagenase (Worthington Biochemical Co., Code CLS, 0-03 %
in Tyrode's solution for 1 h at 37 °C). After isolation, the tissue fragments were
washed thoroughly in serum-supplemented Tyrode's solution, then in Tyrode's
solution. There was no difference between the differentiation of homotypic
recombinants obtained by the use of collagenase and that of intact fragments of
5-day digestive organs. The endoderm of the 3- and 5-day area vitellina was
isolated without collagenase.
Organ culture of tissue fragments in vitro
The endoderm of the yolk sac was cultured for 2 to 21 days in combination
with or without mesenchymes of the digestive tract on an agar clot contained in
embryological watch glasses at 37 °C by the methods of Wolff & Haffen (1952).
The culture medium consisted of seven parts of 1 % bactoagar (Difco) in Gey's
solution, three parts of horse serum (Flow Laboratories), three parts of 12-day
digestive-tract-free chick embryo extract (50 % in Tyrode's solution). Penicillin G
was added. Preliminary experiments showed that the composition of this
medium was best for the differentiation of the yolk-sac endoderm. Some endodermal pieces were cultured in fragments of the vitelline membrane according
to Wolff (1961) and Mizuno & Sumiya (1974).
The explants were transferred on to new culture medium every seventh day.
Intact fragments or homotypic recombinants of 5-day chick digestive organs
were also cultured under the same conditions and served as control.
280
T. MASUI
Differentiation of the yolk-sac endoderm
281
In vivo cultivation of recombinants
After cultivation for 1 to 2 days on the Wolff & Haffen (1952) medium,
recombinants were grafted onto the chorio-allantoic membrane (CAM) of 9-day
chick embryos for a further 7 to 10 days.
Histological methods
After cultivation, the explants were fixed in Bouin's fluid, embedded in
paraffin, and sectioned at 5 fim. The sections were stained with PAS-haematoxylin. Morphological characters of the cell nuclei of chick and quail make the
distinction between the derivation of tissues (Le Douarin, 1969). Some explants
were fixed in Zenker's solution and the sections were stained by the FeulgcnRossenbeck technique (1924).
Analysis of experimental data
The explants were examined according to the following criteria. When various
types of differentiation were formed in one explant, they were all scored.
For the oesophageal epithelium, stratified cuboidal epithelium; for the proventricular epithelium, simple or pseudostratified columnar epithelium and
proventricular glands; for the gizzard epithelium, high pseudostratified columnar
epithelium; for the small intestinal epithelium, simple columnar epithelium with
PAS-positive striated border and the formation of villi in typical cases; for the
yolk-sac parenchymal cells, large round cells with PAS-positive cytoplasm and
large cytoplasmic vacuoles, and often PAS-positive granules.
FIGURES
3-10
In the following recombinations, mesenchyme was obtained from 5-day chick
embryos, and yolk-sac endoderm was obtained from quail embryos.
Fig. 3. Intact EEAP of 5-day quail embryo. The endodermal epithelium (EN) is
very thin, x 670.
Fig. 4. The endoderm of 5-day EEAP alone cultured for 7 days in vitro enveloped in
the vitelline membrane. Undefined cells are seen, x 670.
Fig. 5. The association of 5-day EEAP endoderm and small intestinal mesenchyme
cultured in vitro for 2 weeks. The epithelium is simple columnar, and villi, striated
border, and goblet cells are seen (Small-intestinal-type differentiation), x 670.
Fig. 6. The association of 5-day EEAP endoderm and proventricular mesenchyme
cultured on the CAM for 7 days. Glands are well formed. Mitotic figures are seen
(Proventricular-type differentiation), x 340.
Fig. 7. The association of 5-day EEAP endoderm and proventricular mesenchyme
cultured on the CAM for 10 days. The epithelium is simple columnar, and villi are
well formed (Small-intestinal-type differentiation), x 340.
Fig. 8. The association of 5-day EEAP endoderm and gizzard mesenchyme cultured
on the CAM for 7 days. The epithelium is pseudostratified columnar, and PASpositive granules and secretion are seen (Gizzard-type differentiation), x 670.
Fig. 9. The association of 5-day EEAP endoderm and small intestinal mesenchyme
cultured for ten days on the CAM. The epithelium is simple columnar, and villi are
formed (Small-intestinal-type differentiation), x 340.
Fig. 10. The 5-day quail area vitellina. EC, ectoderm; EN, endoderm. x400.
282
11
T. MASUI
Differentiation of the yolk-sac endoderm
283
RESULTS
1. Self-differentiation potency of the yolk-sac endoderm in vitro
The endoderm of the 5-day EEAP in situ is a simple squamous epithelium
(Fig. 3). When the isolated endoderm was cultured in vitro enveloped in a
fragment of the vitelline membrane, it differentiated mainly into round basophilic cells arranged irregularly (Fig. 4). Long-term cultivation caused necrosis
in the explants, and neither goblet cells nor the striated border differentiated.
The endoderm of the 3- and 5-day area vitellina in situ consists of large cells
swollen with PAS-positive yolk drops and irregularly packed in several layers
(Fig. 10). When cultured directly on the medium, the area vitellina endoderm
differentiated into typical yolk-sac parenchyma (Fig. 11).
The endoderm of the 3- and 5-day area vasculosa in situ consists of differentiated yolk-sac parenchymal cells. In cultures, the endoderm kept its differentiated state for one week, but longer cultivation resulted in necrosis.
FIGURES
11-18
Fig. 11. A part of the endoderm of 5-day area vitellina cultured alone in vitro for one
week. Yolk-sac parenchymal cells differentiated, x 670.
Fig: 12. The association of 3-day area vitellina endoderm and oesophageal mesenchyme cultured for 2 days in vitro. Yolk-sac parenchymal cells developed, x 340.
Fig. 13. The association of 3-day area vitellina endoderm and proventricular
mesenchyme cultured for 3 weeks in vitro. The epithelium is simple columnar, and
typical glands and goblet cells are seen (Proventricular-type differentiation), x 340.
Fig. 14. The association of 5-day area vitellina endoderm and proventricular
mesenchyme cultured for 2 weeks in vitro. All the epithelial cells are simple columnar
with striated border. Glands are also well formed. Goblet cells appear, x 340.
Fig. 15. The association of 5-day area vitellina endoderm and proventricular
mesenchyme cultured for 2 weeks in vitro. Undulated, simple columnar epithelium,
well developed striated border, and goblet cells are seen (Small-intestinal-type
differentiation), x 340.
Fig. 16. The association of 5-day area vitellina endoderm and gizzard mesenchyme
cultured for 2 weeks in vitro. Simple columnar epithelium composed of well developed
yolk-sac parenchymal cells, x 670.
Fig. 17. The association of 5-day area vitellina endoderm and small intestinal
mesenchyme cultured for 3 weeks in vitro. High simple columnar epithelium, well
developed striated border, and goblet cells are seen (Small-intestinal-type differentiation). x670.
Fig. 18. The association of 5-day area vitellina endoderm and oesophageal mesenchyme cultured for 10 days on the CAM. The epithelium is pluristratified. There also
differentiated striated border and goblet cells (Oesophageal-type differentiation).
x670.
284
T. MASUI
Table 1. Differentiation of yolk-sac endoderm cultured on CAM m
association with 5-day digestive-tract mesenchymes
Origin
of
endoderm
5-day EEAP
3- and 5-day
area vitellina
Number of
explants
mesenchyme recovered
Origin
of
OE
PRO
GIZ
SI
OE
PRO
GIZ
SI
16
14
14
15
23
18
17
13
Surface epithelium
X
PRO
GIZ
SI
Villi
Striated
border
PnWft
OE
9
0
0
0
20
3
3
0
5
13
0
1
2
15
0
0
1
4
9
1
0
2
8
2
14
8
9
14
21
13
13
12
8
8
2
14
9
13
1
10
10
0
2
6
3
1
1
4
15
14
6
9
12
10
9
9
f
cells
Abbreviations as Fig. 1.
2. The differentiation of yolk-sac endoderm under the influence of digestivetract mesenchymes
2.1. Differentiation of the EEAP endoderm
When endoderm of the 5-day EEAP was cultured in vitro on mesenchyme of
5-day proventriculus or small intestine, it differentiated into the mesenchymespecific epithelium, and intestinal-type epithelium also developed in all types of
recombinations. Goblet cells appeared after two weeks of cultivation. Villi and
the striated border differentiated in the recombination with small intestinal
mesenchyme after two weeks (Fig. 5). Yolk-sac parenchyma was developed in
combination with the gizzard mesenchyme, while it scarcely appeared in the
other types of recombinations.
When similar recombinants were cultured on the CAM, the mesenchymespecific induction and the intestinal differentiation with villi formation took
place (Table 1, Figs 6-9). Striated border was developed best in association with
the oesophageal mesenchyme. Villus formation and the differentiation of
striated border seemed to occur independently. No yolk-sac parenchyma
differentiated.
2.2. Differentiation of the area vitellina endoderm
The endoderm of the 3- and 5-day area vitellina was cultured in vitro in
association with 5-day digestive-tract mesenchymes. After 2 days of culture, the
endodermal cells differentiated into yolk-sac parenchyma (Fig. 12). Cultured
longer, they became basophilic and proliferative. These basophilic cells differentiated not only into the mesenchyme-specific epithelia under the influence of
the digestive-tract mesenchymes (Table 2, Figs 13,17), but also into epithelium
of small intestinal type (Figs 14, 15, 17). When the explants were cultured for
more than two weeks, a PAS-positive striated border often developed (Figs 14,
Differentiation
of the yolk-sac
285
endoderm
Table 2. Differentiation of 3- and 5-day vitellina endoderm cultured
in vitro in association with 5-day digestive-tract mesenchymes
Origin
of
mesenchyme
OE
PRO
GIZ
SI
Culture
period
(days)
Number of
explants
recovered
7-8
14
21
7-8
14
21
7-8
14
21
7-8
14
21
21
19
27
32
23
41
38
24
45
28
27
42
Surface epithelium
A
OE
PRO
GIZ
1
9
6
0
0
0
0
0
0
0
0
0
0
0
0
14
14
12
0
4
0
0
1
1
7
9
0
7
4
21
0
0
1
0
4
9
SI
10
12
14(1)
11
12(8)
25(11)
6
21(6)
35 (15)
3
15(2)
21(7)
YSP
15
8
4
28
3
2
38
16
4
28
14
3
Goblet
cells
0
6
12
0
20
33
0
23
41
0
26
32
Numbers in parentheses indicate the number of explants differentiated into intestinal epithelium with
striated border.
Abbreviations as Fig. 1. YSP: yolk-sac parenchyma.
15, 17), and it was as high as that observed in the intact small intestine cultured
in vitro. Goblet cells appeared in all types of recombinations. The results
are summarized in Fig. 2.
When the endoderm of the area vitellina of 3- or 5-day embryos was cultured
on the CAM in combination with the digestive-tract mesenchymes, the results
were similar to those obtained in vitro (Table 1). The endoderm of the area
vitellina differentiated into mesenchyme-specific epithelia and also into intestinal
type epithelium with goblet cells and well-developed villi in all types of combinations. In association with the oesophageal mesenchyme, striated border developed on a stratified cuboidal epithelium (Fig. 18).
2.3. Differentiation of the area vasculosa endoderm in vitro
Endoderm of the 3- and 5-day area vasculosa was cultured in vitro in association with 5-day digestive-tract mesenchymes. The endoderm became necrotic
and scarcely differentiated into mesenchyme-specific epithelia for intestinal-type
epithelium.
DISCUSSION
Self-differentiation of yolk-sac endoderm
In normal development, the area vitellina endoderm differentiates into yolksac parenchyma according to the expansion of the mesoderm (Bellairs, 1963;
Bennett, Dubois & Chapeville, 1972; Bennett, 1973; Masui, 1978). The present
10
EMB 62
286
T. MASUI
investigation clearly demonstrated that isolated endoderm of the area vitellina
can self-differentiate into yolk-sac parenchyma in the absence of mesenchyme,
when the endoderm was cultured alone in vitro. Moreover, even when cultured
with inductively active mesenchymes of the digestive-tract, the area vitellina
endoderm differentiated into yolk-sac parenchyma in the first place. Therefore,
it is conceivable that the area vitellina endoderm has an intense potency to
differentiate into the yolk-sac parenchyma by itself.
In contrast, the EEAP endoderm hardly differentiated into yolk-sac parenchyma in normal development (Masui, 1978) and when cultured alone in vitro
(in the present study). It has been reported, however, that area pellucida endoderm of early stages can self-differentiate into yolk-sac parenchyma when it is
cultured in vitro enveloped in the vitelline membrane (Sumiya, 1976). In the
present investigation, it was demonstrated that the EEAP endoderm differentiated into yolk-sac parenchyma when cultured with gizzard mesenchyme.
It might be that the EEAP endoderm can barely self-differentiate alone into
yolk-sac parenchyma, but still has that potency, and, in adequate conditions
can differentiate into yolk-sac parenchyma.
Inductive ability of digestive-tract mesenchymes and the
differentiation potency of yolk-sac endoderm
Specific induction has been studied between the digestive-tract mesenchymes
and heterologous epithelia: gizzard or proventricular mesenchyme and epidermis (McLoughlin, 1961), gizzard mesenchyme and proventricular epithelium
(Sigot, 1963; Sigot & Marin, 1970), proventricular or intestinal mesenchyme
and ureter (Bishop-Calame, 1966), proventricular, gizzard, or intestinal mesenchyme and bronchial epithelium (Dameron, 1968), digestive-tract mesenchymes
and allantoic endoderm (Mizuno & Yasugi, 1973; Yasugi & Mizuno, 1974;
Yasugi, 1976a, b, 1979; Gumpel-Pinot et al. 1978), and mesenchymes and
epithelia of digestive tract (Yasugi & Mizuno, 1978; Gumpel-Pinot et al. 1978).
In the present investigation we demonstrated that, when the area vitellina
endoderm was cultured with digestive-tract mesenchymes, the endodermal cells
differentiated into yolk-sac parenchymal cells at first, but later they became
basophilic and proliferative, and lastly they differentiated not only into the
mesenchyme-specific epithelia of the digestive tract but also into small intestinal
type epithelium. The EEAP endoderm, which is originally basophilic, also can
differentiate into mesenchyme-specific epithelia and the small intestinal type
epithelium under the influence of digestive-tract mesenchymes.
The results of the present investigation confirm the instructive induction of
the digestive-tract mesenchymes, and demonstrate clearly the competence of the
area vitellina and EEAP endoderm to the induction by the digestive-tract
mesenchymes.
The endoderm of the area vitellina and EEAP does not differentiate into
intestinal type epithelium with goblet cells or striated border, during normal
Differentiation of the yolk-sac endoderm
287
development (Bellairs, 1963; Bennett et al. 1972; Bennett, 1973; Masui, 1978;
Mobbs & McMillan, 1979) nor in in vitro culture (the present investigation).
However, when cultured with digestive-tract mesenchymes, the yolk-sac endoderm sometimes differentiated into small intestinal type epithelium with goblet
cells, striated border and villi, in all types of recombinations. These results
indicate that the endoderm of the area vitellina and EEAP can manifest its
differentiation potency to intestinal epithelium under non-organ-specific stimuli
of digestive-tract mesenchymes.
In the recombination experiments, it is not conceivable that the mere increase
in cell number by the association with mesenchymes could affect the differentiation pattern of the yolk-sac endoderm, because the yolk-sac endoderm differentiated into mesenchyme-specific epithelia according to the types of combined
digestive-tract mesenchymes. Further, when cultured alone without any mesenchymal cells, the area vitellina endoderm differentiated only into yolk-sac
parenchyma, even if the volume of endoderm in an explant was increased.
The influence of yolk-sac endoderm on digestive-tract mesenchymes
Little is known about the inductive influence of the epithelium to the mesenchyme: the small intestinal epithelium induced villus formation in intrasplanchnopleural grafting (Gumpel-Pinot, et al. 1978) and in the CAM grafting
after recombination with heterologous mesenchymes (Yasugi & Mizuno, 1978).
The present investigation revealed that villi were formed when the area vitellina
or EEAP endoderm was cultured on the CAM with digestive-tract mesenchymes.
The villus formation did not take place unless the epithelium differentiated into
intestinal type epithelium. Therefore the villus formation seems to occur only
when the epithelium manifests intestinal differentiation.
Mechanisms of differentiation of the area vitellina endoderm into mesenchymespecific epithelia and those of the intestinalization under the influence of the
digestive-tract mesenchymes are problems worth further studies. We are now
following the course of the differentiation of the yolk-sac endoderm and giving
attention to the expression of tissue-specific enzymes.
The author wishes to express his deep gratitude to Professor Takeo Mizuno of the University of Tokyo for his valuable advice and encouragement during the course of this work.
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{Received 27 June 1980, revised 30 September 1980)