/. Embryo/, exp. Morph. Vol. 52,. pp 49-62, 1979
Printed in Great Britain © Company of Biologists Limited 1979
49
The development of hepatogenic potency in the
endoderm of quail embryos
By SUMIKO FUKUDA 1
From the Zoological Institute, Faculty of Science,
University of Tokyo
SUMMARY
Hepatogenic potency of the endoderm is detectable in the anterior half of the endoderm of
quail embryos older than 2-somite stage when endodermal fragments are cultured with or
without heterologous chick mesenchymes, in the coelomic cavity of 3-day chick embryos. On
the other hand, the posterior half of the endoderm never has hepatogenic potency. The
hepatogenic potency of the endoderm is gradually stabilised with increasing age. However,
expression of hepatogenesis can be affected when the endoderm is associated with inductively
active digestive tract mesenchymes.
Mesenchyme taken from the presumptive cardiac region ('cardiac' mesenchyme) of chick
embryos is necessary for the uncommitted anterior endoderm to acquire hepatogenic potency,
and this effect is specific for the 'cardiac' mesenchyme. The 'cardiac' mesenchyme, however,
fails to induce hepatic epithelium in the allantoic endoderm, which can differentiate heterotypically when cultured in combination with digestive tract mesenchymes. The evidence presented in this study suggests that the effect of 'cardiac' mesenchyme on the acquisition of
hepatogenic potency in the endoderm is limited.
INTRODUCTION
Embryonic development of the liver involves a complex series of cellular
interactions. In the chick embryo, the primordium of the liver appears around
the 20-somite stage as an endodermal evagination of the floor of the anterior
intestinal portal. The hepatic endoderm proliferates, and together with the
surrounding mesenchymal tissue including the endothelium, finally differentiates
into the hepatic structure composed of hepatic cords and sinusoids (Kingsbury,
Alexanderson & Kornstein, 1956; Romanoff, 1960). We have previously described the normal morphogenesis of the liver of the chick embryo with special
reference to the developmental interrelation of liver elements: the hepatic
endoderm, the hepatic mesenchyme and the endothelium (Fukuda, 1976).
Interactions of these components during the morphogenesis of the liver have
been experimentally analysed (Le Douarin, 1964; Houssaint & Le Douarin,
1971; Sherer, 1975).
1
Author's address: Zoological Institute, Faculty of Science, University of Tokyo, Hongo,
Tokyo, 113 Japan.
50
S. FUKUDA
Le Douarin (1964, 1975) showed that the endoderm becomes capable of
differentiating into hepatic epithelium when associated with the hepatic mesenchyme ('determination' of hepatic endoderm) at the 4- to 5-somite stage far
earlier than the initial hepatic evagination, and that the 'determination' of the
hepatic endoderm depends on the presence of the pre-cardiac mesenchyme.
The importance of the cardiac mesenchyme in hepatic differentiation has been
indicated by Hunt (1931), Willier & Rawles (1931) and Rawles (1936).
Recent experiments with the chick embryo (Sumiya, 1976; Sumiya & Mizuno>
1976; Mizuno & Sumiya, 1977) have established that anterior fragments of the
endoderm older than the 7-somite stage can differentiate into hepatic epithelium
in vitro in the absence of mesenchymal cells.
This paper deals with the developmental processes of the hepatic endoderm
before hepatic primordium formation in quail embryos. The developmental
interrelation of the liver elements before the initial hepatic evagination is described, and the first appearance of endoderm possessing hepatogenic potency is
clarified. The degree of hepatogenic potency in endoderm from various developmental stages, when cultured in association with heterologous mesenchymes, is defined. Some mesenchymes from the digestive tract have been
demonstrated to have inductive ability (Sigot, 1971; Mizuno & Yasugi, 1973).
Finally, the specificity and effect of the 'cardiac' mesenchyme on the aquisition
of hepatogenic potency in the endoderm is demonstrated: the 'cardiac' mesenchyme is associated with the anterior endoderm or the allantoic endoderm,
which has been proved to differentiate heterotypically under the influence of
inductively active digestive-tract mesenchymes (Mizuno & Yasugi, 1973;
Yasugi & Mizuno, 1974; Yasugi, 1976a, b; Gumpel-Pinot, Yasugi & Mizuno,
1978).
MATERIALS AND METHODS
Embryos
Japanese quail (Coturnix coturnix japonica) and White Leghorn (Gal/us
gal/us domesticus) embryos were used. The stages were identified by the appearance of blastoderms or by the number of paired somites for younger embryos,
and by days of incubation for older embryos.
To exclude cellular contamination of either of the tissue components, the
difference of nuclear structure between chick and quail cells was used as a
biological cell marker (Le Douarin, 1969). Endodermal tissues were obtained
from quail embryos and mesenchymal tissues from chick embryos.
Isolation of endodermal fragments
Quail embryos from definitive streak to 21-somite stage were cut into two
pieces (the anterior and posterior pieces) at various antero-posterior levels
according to the developmental stages: for pre-somite stages, the level was
posterior to the posterior end of Hensen's node; for 1- to 4-somite stage,.
Hepatogenic potency in endoderm of quail embryos
51
posterior to the last somite; for older than 5-somite stage, between the 4th and
the 10th somite. These anterior and posterior pieces were treated in 003 % collagenase (Worthington, CLSPA) in Tyrode's solution for 80 min at 37 °C5 and
anterior and posterior endodermal fragments were isolated. The areas of secure
adhesion between the endodermal layer and overlying tissues, the area under the
anterior half of the primitive streak and the head process and the apex of the
foregut, were excluded from the preparation of endodermal fragments. Allantoic endoderm was obtained from the allantois of 3-day-old quail embryos.
These endodermal fragments were washed thoroughly in three changes of
serum-supplemented Tyrode's solution to eliminate the enzyme, and finally in
fresh Tyrode's solution.
Preparation of mesenchymes
The mesenchymes of several definite parts of the digestive tract (oesophagus,
proventriculus, gizzard and small intestine), were obtained from 5- to 15-day
chick embryos by collagenase treatment.
'Cardiac' mesenchyme was obtained from the presumptive cardiac and the
cardiac region (Rudnick, 1938; Rawles, 1943; Le Dourain, 1964) of head fold
to 11-somite-stage chick embryos with the aid of collagenase. Non-cardiac
mesenchyme was obtained from the area posterior to the 4th somite for 4- to
6-somite-stage embryos and posterior to the 5th to 7th somite for 7- to 11somite-stage embryos.
In vitro association of tissue fragments
The endodermal fragments or allantoic endoderm of quail embryos were
cultured in interspecific combination with various heterologous (non-hepatic),
'cardiac', or non-cardiac mesenchymes for 1 day in vitro according to the modified method of Wolff & HafFen (1952): the culture medium consisted of seven
parts of 1 % Difco Bacto-Agar in Gey's solution, three parts of foetal calf serum
(Flow Laboratories Ltd.), three parts of medium 199 containing Earle's
balanced salt solution (Morgan, Morton & Parker, 1950) and one part of
Tyrode's solution containing potassium penicillin G.
In vivo cultivation of endodermal fragments and recombinants
Isolated anterior and posterior endodermal fragments were transplanted
directly into the coelomic cavity of 3-day chick embryos and cultured for 6-8
days.
Interspecific quail-chick recombinants of various tissue fragments cultured
in vitro were transplanted into the coelomic cavity or grafted onto the chorioallantoic membrane, and were harvested after 6-8 days.
Histological methods
The explants were fixed in Bouin's fluid, embedded in paraffin, and sectioned
at 5/Am. The sections were stained with Carazzi's haematoxylin and eosin. To
detect glycogen, PAS-staining was carried out after fixation in Gendre's fluid.
52
S. FUKUDA
Table 1. Sequence of development of liver elements
Stages
Splanchnic
mesenchyme
Embryonic
endothelium
Hepatic
mesenchyme
Definitive streak
Head process
Head fold
1-2 somite
3-4 somite
5-6 somite
7-8 somite
9-10 somite
11-13 somite
14-16 somite
17-19 somite
20-22 somite
23-25 somite
26-28 somite
29-32 somite
—
—
—
+
+
+
+
+
+
+
+
+
+
+
—
—
—
—
—
—
+
+
+
+
+
+
+
+
+
+
—
—
—
—
—
+
+
+
+
Criteria for identifying the differentiation of hepatic epithelium
The formation of bile canaliculi characteristically bordered by several hepatic
cslls and of hepatic cords composed of two rows of hepatocytes identifies the
hepatic epithelium. Glycogen storage in cytoplasm of hepatic cells and the
presence of intrahepatic bile ducts composed of cuboidal epithelial cells with
homogeneous cytoplasm are also standards for the identification of hepatic
epithelium.
RESULTS
Development of three major tissue components related to the differentiation of the
liver
The early development of the hepatic endoderm, the hepatic mesenchyme and
the embryonic endothelium of quail and chick embryos was studied with
special reference to their temporal and spatial interrelations. The results with
the quail and chick embryos were basically similar and are summarized in
Table 1 and Fig. 1.
Around the 20-somite stage the endoderm of the margin of the anterior
intestinal portal evaginates and forms the hepatic primordium. At this time, the
hepatic endoderm is in close contact with the endothelium of the omphalomesenteric vein and splanchnic mesenchyme.
Splanchnic mesenchyme develops at the latest by the 4-somite stage, and
contacts with the endoderm of the anterior intestinal portal. The splanchnic
mesenchyme develops into the cardiac mesenchyme, endothelium and a loose
mesenchymal tissue.
Hepatogenic potency in endoderm of quail embryos
Stages
53
Hepatic
endoderm
14-16 somite
17—19 somite
20—22 somite
23-25 somite
26-32 somite
Fig. 1. Schematic drawings of the formation and branching of hepatic primordial
endoderm.
The loose mesenchyrnal tissue develops around the ductus venosus somewhat
after the onset of hepatic-bud evagination. The hepatic primordial endoderm
and the endothelial cells proliferate and invade the loose mesenchymal tissue,
which is identical to the hepatic mesenchyme, and form hepatic cords and
sinusoids.
The endothelium of cardiac and anterior intestinal portal region first appears
at the 5- to 6-somite stage, much earlier than the beginning of the hepatic-bud
evagination.
Anterior and posterior endodermal fragments cultured alone in the coelomic
cavity
Anterior and posterior endodermal fragments isolated from various stages of
quail embryos were transplanted into the coelomic cavity of 3-day chick
embryos (Fig. 2). After 6-8 days' cultivation, about two-thirds of explants could
be found to attach loosely to various mesenchymes of the host, adjacent to the
coelomic cavity, or to float in the abdominal cavity forming cyst-like structures.
Histological sections showed that the endodermal epithelium consisted of
quail cells and the mesenchyme, chick cells. As shown in Table 2, the hepatogenic
potency of the anterior endoderm can first be demonstrated at the 5-somite
stage in these experimental conditions (Fig. 5), and the frequency of hepatic
epithelium formation becomes higher as development proceeds. After the
onset of hepatic morphogenesis (as shown by * in Table 2) the incidence becomes about 84 %. The hepatic epithelium never develops from the posterior
endodermal fragments regardless of stage. Besides the hepatic epithelium,
54
S. FUKUDA
Anterior endoderm
Definitive streak
to 21-somite stage
quail embryo
3-day chick embryo
Fig. 2. Schematic representation showing the isolation of anterior and posterior
endodermal fragments from definitive streak to 2.1 -somite-stage quail embryos.
These endodermal fragments alone are transplanted into the coelomic cavity of
3-day chick embryo. AIP, anterior intestinal portal.
Table 2. Areas and stages of the quail endoderm from which hepatic epithelium
differentiated, when the endoderm was cultured in the coelomic cavity of chick
embryos
Stages
Definitive streak
Head process
Head fold
1-2 somite
3-4 somite
5-6 somite
7-8 somite
9—10 somite
11-12 somite
13-14 somite
15-21 somite
21-35 somite
Anterior endoderm
0/3
0/9
0/7
0/9
0/8
5/22
4/16
7/18
3/9
4/7
3/6
26/31
(0)\
(0)
(0)
(0)
Posterior endoderm
0/16(0)
(oy
(25)
(38)
(33)
(57)
(50)
0/20 (0)
* (84)J
No. in parentheses designates the percentage of differentiation of the hepatic epithelium.
* Hepatic primordial endoderm was cultured.
oesophageal, proventricular, gizzard, intestinal, pancreatic and yolk-sac
epithelia-like structures often developed in the explants.
It can be concluded from these results that the endoderm possesses hepatogenic potency at the latest by the 5-somite stage in quail embryos, and that
this potency is restricted to the anterior half of the endoderm, and becomes
gradually stable as development proceeds.
Combination of anterior endoderm and heterologous mesenchyme
To define the stability of the differentiation capacity of the hepatic endoderm,
anterior endodermal fragments derived from definitive streak to 19-somite-
Hepatogenic potency in endoderm of quail embryos
55
Series A
Definitive streak
to 19-somite stage
quail embryo
Coelomic
implantation
Digestive traet
of 8-to 15-day
chick embryo
Mesencliyme
Series B
Definitive streak
to 1 1-somite stage
quail embryo
Cliorio-allantoic
membrane grafts
Digestive tract
of 5-day chick
embryo
Inductively active
mesencliyme
Fig. 3. Diagram showing the two series of combinations of quail anterior
endoderm with various chick mssenchymes.
Table 3. Differentiation of hepatic epithelium from anterior endoderm associated
with heterologous mesenchymes
Stages of
the endoderm
Series A
Series B
Definitive streak
Head process
Head fold
1-2 somite
3-4 somite
5-6 somite
7-8 somite
9—10 somite
11-12 somite
13-19 somite
0/4
0/4
0/9
1/16
2/13
2/5
1/4
0/6
2/6
2/4
0/4
0/14
0/10
0/23
1/32
4/44
1/27
4/8
1/3
1/1
stage quail embryos were associated with heterologous mesenchymes of the
digestive tract of chick embryos. Two series of experiments were prepared
(Fig. 3): series A, the anterior endoderm was combined with the mesenchymes
of 8- to 15-day digestive tract; series B, the anterior endoderm was combined
with mesenchymes of 5-day digestive tract.
In series A, we found that oesophageal, proventricular, and small intestinal
mesenchymes of 8- to 14-day embryos permitted differentiation of hepatic
56
S.
FUKUDA
epithelium from the anterior endoderm of embryos older than 2-somite stage
(10 cases out of 46) (Fig. 6 and Table 3).
In series B, hepatic epithelium differentiated less often (12 cases out of 125).
The differentiation of hepatic epithelium was limited to endoderm older than
the 4-somite stage (Table 3).
The results of these two series of experiments indicate that the anterior endo­
derm possesses hepatogenic potency at the earliest from the 2-somite stage or
at the latest from the 4-somite stage, and that this potency is expressed even in
the presence of heterologous mesenchymes, although the expression of hepatogenesis by the anterior endoderm can be affected, especially when associated
with inductively active mesenchymes of 5-day digestive tract (series B).
Specificity and effect ofcardiac'
mesenchyme in the development of the liver
The specificity and effect of mesenchyme taken from the presumptive cardiac
region ('cardiac' mesenchyme) in the acquisition of the differentiation potency
of the hepatic endoderm in quail embryos were investigated. The experimental
procedure is shown in Fig. 4. The anterior endodermal fragments from definitive
streak to 1-somite-stage embryos were associated with 'cardiac' or non-cardiac
mesenchyme. The recombinants were cultured in the coelomic cavity or on the
chorio-allantoic membrane. To test the inducing effect of the 'cardiac' mesen­
chyme, allantoic endoderm of 3-day quail embryos was also combined with
these mesenchymes and cultured in the coelomic cavity.
Hepatic epithelial cells with heavy glycogen storage in their cytoplasm, and
forming hepatic cords and bile canaliculi often differentiate from the anterior
endoderm associated with the 'cardiac' mesenchyme. (Fig. 7). Most of these
explants were accompanied by rhythmically pulsating cardiac tissue. In con­
trast, the anterior endodermal fragments cultured with non-cardiac mesenchyme
seldom differentiated into hepatic epithelium and mostly remained undifferen­
tiated (Table 4). The allantoic endoderm associated with the 'cardiac' or noncardiac mesenchyme, never differentiated into hepatic epithelium even when
pulsating cardiac tissue differentiated, and remained undifferentiated (Fig. 8).
These results strongly suggest specific dependence on 'cardiac' mesenchyme
of the differentiation of hepatic epithelium in quail embryos as has been sug­
gested in chick embryos. However, 'cardiac' mesenchyme fails to induce hepatic
epithelium in the allantoic endoderm, which possesses considerable ability to
undergo heterotypic differentiation. Other types of epithelia such as intestine­
like epithelium often differentiate in the allantoic endoderm associated with
'cardiac' as well as non-cardiac mesenchymes.
DISCUSSION
The hepatogenic potency of endoderm has been investigated extensively in the
chick embryo. The endoderm of the presumptive hepatic area taken from
embryos older than 4-somite stage can differentiate into hepatic epithelium only
Hepatogenic potency in endoderm of quail embryos
57
3-ilav q u a i l e m b r v o
Coelomic
implantation
Fig. 4. Diagram showing the mode of association of quail anterior or allantoic
endoderm with chick 'cardiac' or non-cardiac mesenchyme.
Table 4. Differentiation of the anterior endoderm of definitive streak to 1-somite
stage or allantoic endoderm under the influence of 'cardiac' or non-cardiac
mesenchyme
' Cardiac '
mesenchyme
Non-cardiac
mesenchyme
Culture
condition
Anterior
Anterior
9/10
20/27
2/13
0/36
Allantoic
0/17
0/7
Coelomic cavity
Chorio-allantoic
membrane
Coelomic cavity
Origin of
the endoderm
under the influence of hepatic mesenchyme (Le Douarin, 1964). However,
foregut endoderm older than the 7-somite stage can differentiate into hepatic
epithelium when it is associated with lateral plate mesenchyme (Le Douarin &
Bussonnet, 1966; Le Douarin, Bussonnet & Chaumont, 1968).
The present investigation revealed that the hepatogenic potency of the
anterior endoderm of the quail embryo appeared between the 2- and 5-somite
stages, when the endoderm was cultured in the host chick, with or without
various heterologous mesenchymes. Though these stages vary a little according
to the experimental conditions (Tables 2, 3), the first appearance of hepatogenic
potency precedes the appearance of hepatic primordia and hepatic mesenchyme.
These stages almost coincide with the stages when the endoderm becomes
capable of differentiating to hepatic epithelium under the influence of hepatic
mesenchyme.
58
S. FUKUDA
Hepatogenic potency in endoderm of quail embryos
59
It has been reported that the hepatic primordial endoderm can never express
its hepatogenic potency, if introduced into the coelomic cavity of a 3-day host
(Le Douarin, 1964, 1975). However, in the present study of more than 200
endodermal fragments transplanted into the coelomic cavity before development of hepatic primordia, two-thirds survived and differentiated into hepatic
epithelium as well as many kinds of digestive tract epithelia. As the explants
were loosely attached to the mesenchymes adjacent to the coelomic cavity, or
were floating in the abdominal cavity, they seemed to be under minimal influence of the mesenchymes and to express their own differentiation potency,
that is, for self-differentiation.
Sumiya (1976), Sumiya & Mizuno (1976) and Mizuno & Sumiya (1977)
demonstrated that endodermal fragments taken from embryos older than the
7-somite stage can differentiate into hepatic epithelial cells when foregut endodermal fragments are cultured in vitro enveloped in a fragment of the vitelline
membrane, but in the absence of any type of mesenchyme. These results,
together with those obtained in the present study, indicate that the foregut
endoderm at these developmental stages has been committed towards hepatic
development.
It must be noted that the time of appearance of self-differentiation potency in
the endoderm for the hepatic epithelium, coincides with that of the first differentiation of embryonic endothelium in the anterior intestinal portal region. The
endothelium of this region is generally considered as the most important in
hepatic morphogenesis (Kingsbury, Alexanderson & Kornstein, 1956; Karrer,
1961; Le Douarin, 1964; Sherer, 1975; Fukuda, 1976).
Data from the present investigation show that the expression of the potency
to differentiate into hepatic endoderm is often prevented by inductively active
heterologous mesenchymes: the differentiation of hepatic epithelium from
endodermal fragments was strongly prevented by association with 5-day
digestive tract mesenchymes. The regionally specific inductive ability of these
mesenchymes has been demonstrated (Sigot, 1971; Mizuno & Yasugi, 1973).
F I G U R E S 5-8
Fig. 5. Quail anterior endoderm of 6-somite stage cultured in the coelomic cavity of
a 3-day chick embryo for 8 days. Differentiation of hepatic epithelium forming
hepatic cords and bile canaliculi.
Fig. 6. Quail anterior endoderm of 4-somite stage cultured in combination with
small intestinal mesenchyme of a 8-day chick embryo for 8 days in the coelomic
cavity. Differentiation of hepatic cords and bile canaliculi.
Fig. 7. Quail anterior endoderm at head-fold stage cultured in combination with
chick 'cardiac' mesenchyme of 6-somite stage. Hepatic tissue with hepatic cords and
bile canaliculi develops well after 8 days' cultivation in the coelomic cavity.
Fig. 8. Quail allantoic endoderm (3-day) cultured combined with chick 'cardiac'
mesenchyme of 4-somite stage. The endoderm remains as undifferentiated cellular
masses or sheets.
60
S. FUKUDA
In the present study, other types of epithelia such as oesophageal, proventricular, gizzard, intestinal, were frequently observed to differentiate in the endoderm associated with the mesenchymes of these organs.
The differentiation potency of hepatic primordial endoderm in heterologous
combination (Le Douarin, 1964; Houssaint & Le Douarin, 1971; Sherer, 1975)
or in coelomic culture (Fukuda & Mizuno, 1978) has been investigated. According to our work, the hepatic primordial endoderm differentiates into hepatic
epithelium and further into the epithelia of intra- and extrahepatic bile ducts and
gall bladder in coelomic cultures. These facts, together with the results obtained
in the present study, indicate that the hepatogenic potency of the hepatic endoderm is stabilized at the stage when the hepatic primordium forms.
The importance of cardiac mesenchyme in hepatic differentiation has been
indicated previously (Hunt, 1931; Willier & Rawles, 1931; Rawles, 1936).
According to these authors, liver differentiation is always accompanied by
differentiation of cardiac tissue. Le Douarin (1964, 1975) reported that the
presence of pre-cardiac mesoderm is necessary for 'determination' of hepatic
endoderm. The present study also demonstrates the necessity and specificity of
the 'cardiac' mesenchyme for the aquisition of hepatogenic potency in the uncommitted endoderm, by the results obtained from experimental combinations
between endoderm and 'cardiac' or non-cardiac mesenchyme (Table 4).
Though the 'cardiac' mesenchyme used in the present study was taken from the
presumptive cardiac region or the cardiac region, it is conceivable that it contained mesenchymal cells which may participate in the formation of organs
other than the heart. What kinds of cell population in mesenchyme taken from
the presumptive cardiac region are really responsible for the acquisition of the
hepatogenic potency, and whether there is a causal relation between the evolution of hepatic endoderm and the cardiac differentiation are unreselved.
Allantoic endoderm, which is known to have considerable ability for heterotypic differentiation (Mizuno & Yasugi, 1973; Yasugi & Mizuno, 1974;
Yasugi, 1976a, b; Gumpel-Pinot et al. 1978), did not acquire hepatogenic
potency when exposed to 'cardiac' mesenchyme. Our experimental data have also
shown that the 'cardiac' mesenchyme does not affect the posterior part or area
opaca of definitive streak to head-fold-stage blastoderms (Fukuda, unpublished).
These results indicate that the inducing ability of the 'cardiac' mesenchyme is
limited. The distribution of endoderm responsive to stimulation by 'cardiac'
mesenchyme will be described in a separate communication.
The author wishes to express her deep gratitude to Professor Takeo Mizuno of the University of Tokyo for his valuable advice and encouragement during the course of this work. She
is also grateful to Doctor Madeleine Gumpel-Pinot of Institut d'Embryologie du CNRS,
Nogent-sur-Marne, France, for her kind instruction in the technique of intracoelomic grafts.
This work was partly supported by Grants from the Ministry of Education, Science and
Culture, Japan to Professor Mizuno.
Hepatogenic potency in endoderm of quail embryos
61
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