J. Embryol. exp. Morph. Vol. 62, pp. 139-152, 1981
Printed in Great Britain © Company of Biologists Limited 1981
139
Enzymo- and immunocytochemical
analyses of the differentiation of liver cells in
the prenatal mouse
ByNOBUYOSHI SHIOJIRI1
From the Zoological Institute, Faculty of Science, University of Tokyo
SUMMARY
Differentiation of the endodermal cells of the mouse liver was studied enzymo- and
immunocytochemically by analyzing the cellular localization of alphafoetoprotein (AFP),
glycogen, and alkaline phosphatase (ALP) and 5'-nucleotidase (5'-Nase) activities.
1. In 8-5-day foetuses, AFP appears in some endodermal cells of the anterior intestinal
portal region. The cells of the cranial diverticulum contain much AFP at 9-5 days, while those
of the caudal diverticulum contain less AFP.
2. In 9-5- to 15-5-day foetuses, hepatocytes are intensely fluorescent for AFP. After
16-5 days less-positive hepatocytes increase in number. AFP is still present in a few hepatocytes of 14-day-old postnatal mice. ALP and 5'-Nase activities appear in a small proportion
of hepatocytes at 13-5 and 14-5 days of embryonal life, respectively. At 15-5 days, many
hepatocytes possess these enzyme activities, and initiate accumulation of glycogen. AFPcontaining hepatocytes type I (gestation day 9-5-16-5) successively acquire ALP and 5'-Nase
activities and accumulate glycogen, and then differentiate into hepatocytes type II after
gestation day 17-5.
3. Endodermal cells constituting lumen structures in the liver trabeculae are the precursor
of the intrahepatic bile duct cells. They possess much AFP, but no glycogen and no ALP
activity, and are similar to hepatocytes type I. Since immature hepatic duct cells also possess
much AFP, but no glycogen, and no ALP and 5'-Nase activities, they are similar to endodermal cells of the lumen structures. Therefore, that the endodermal cells of the lumen
structures are the intermediate cells between hepatocytes type I and hepatic duct cells may be
conceivable.
INTRODUCTION
In mammalian embryos, hepatocytes differentiate as cell cords extended from
the stratified hepatic endoderm (Wilson, Groat & Leduc, 1963; Severn, 1972;
Shiojiri, 1979). Purtilo & Yunis (1971) reported that AFP, a product of embryonal hepatocytes, is present in the hepatic bud of human embryos. However,
all cells constituting the hepatic primordium do not necessarily differentiate
into hepatocytes. Therefore, two of the problems to be resolved might be
whether AFP is present only in the presumptive hepatocytes, and how the
1
Author's address: Zoological Institute, Faculty of Science, University of Tokyo, Hongo,
Tokyo 113, Japan.
140
N. SHIOJIRI
morphological and functional differentiation of hepatocytes is related to the
synthesis of AFP.
It has been well known that drastic morphological differentiation and enzymic
differentiation occur in hepatocytes during perinatal life of mammals (Greengard, 1969; Greengard, Federman & Knox, 1972; Chedid & Nair, 1974;
Shiojiri, 1979), but few studies have been done on the development of ALP and
5'-Nase activities in the bile canaliculi of hepatocytes (Turchini, 1961; De WolfPeeters, De Vos & Desmet, 1972).
As to the development of the intrahepatic bile ducts, two main theories have
been advocated. One is that the intrahepatic bile ducts develop from the heads
of the hepatic ducts (Hammer, 1926; Koga, 1971). The other is that the intrahepatic bile ducts originate from hepatocytes (Bloom, 1926; Wilson et ah 1963;
Enzan, Ohkita, Fujita & Iijima, 1974). We have demonstrated previously that,
in mouse embryos, the precursor cells of the intrahepatic bile duct cells are
similar to both immature hepatocytes and hepatic duct cells, and that the
precursor cells are situated between the immature hepatocytes and hepatic duct
cells and connect with both of them (Shiojiri, 1979).
Hepatocytes store glycogen in the cytoplasm, and ALP and 5'-Nase activities
are demonstrated in the bile canaliculi (Wachstein & Meisel, 1957; Wachstein,
1959). However, glycogen and these enzyme activities are not demonstrated in
the epithelial cells of the intrahepatic bile ducts. To clarify the processes of the
formation of the intrahepatic bile ducts, it might be effectual to compare these
cytochemical characteristics of the hepatocytes, hepatic duct cells and precursor cells of the intrahepatic bile duct cells in the developing liver.
The purpose of the present investigation is to clarify the differentiation of
hepatocytes and biliary duct cells by describing the localization of AFP,
glycogen, and ALP and 5'-Nase activities during the development of the mouse
liver.
MATERIAL AND METHODS
Animals
ICR strain mice (CLEA Japan, Tokyo) were used. The animals were mated
during the night and copulation was confirmed by the presence of the vaginal
plug the next morning. The conceptus was considered 0-5 days at 12 noon of
this day.
Immunefluorescent method for AFP
Tissues were fixed in a mixture of 96 % ethanol and glacial acetic acid (99:1 v/v)
at 0 °C for 12 to 24 h according to Engelhardt, Goussev, Shipova & Abelev
(1971). The tissues were dehydrated in two changes of cold ethanol, cleared
through three changes of cold xylene, and embedded in paraffin at 53 °C. Serial
sections were cut at 5 fim thickness and slides dried for 1 h at 37 °C. Sections
were washed in two baths of 0-01 M-phosphate-buffered saline (PBS, pH 7-2)
Cytochemical differentiation of mouse liver cells
141
for 5 min before application of antisera. Sections were incubated with rabbit
antisera against mouse AFP (Miles Lab., Inc., Indiana) at 1/40 dilution for
1 h at room temperature, washed thoroughly with PBS, incubated with FITCconjugated goat anti-rabbit IgG antibodies (Miles Lab., Inc., Indiana) at 1/64
dilution for 1 h, washed again, mounted in buffered glycerol.
Two control incubations were done by (1) treatment, with anti-AFP antisera
without FITC-conjugated goat anti-rabbit IgG antibodies, and (2) incubation
only with FITC-conjugated goat anti-rabbit IgG antibodies.
The sections were examined with an Olympus fluorescent microscope (model
BHF) and photographed with Kodak Tri-X pan film.
Staining method for localization of ALP activity
Tissues were fixed in cold 100 % ethanol for 24 h, cleared through two changes
of cold xylene and embedded in paraffin at 53 °C. Sections were cut at 5 [im
thickness and slides dried for 1 h at 37 °C. Incubations (pH 9-6) were performed
for 1 h at 37 °C according to Gomori (1939). Control experiments were carried
out by (1) incubation without the substrate, and (2) inhibition with L-cysteine.
The sections were kept in a 1 mM solution of L-cysteine for 10 min before being
incubated in a standard medium with 1 mM L-cysteine.
Staining method for localization of 5'-Nase activity
Tissue specimens for 5'-Nase activity were frozen in hexane cooled by dry iceethanol. Frozen sections were cut at 6 fim thickness on a cryostat, mounted on
a slide glass and fixed in ice-cold ethanol for 10 min. Incubations were carried
out for 1 h at 37 °C in Wachstein and Meisel (1957) medium. The medium contained 1 mM 5'-adenylic acid, 10 mM magnesium sulphate and 3-6 mM lead
nitrate, dissolved in 0-1 M-tris-maleate buffer, pH 7-2. Control sections were
incubated in the medium without the substrate. To exclude nonspecific ALP
activity, incubations were carried out with sodium /?-glycerophosphate as substrate in the same concentration as AMP. After incubation, the sections were
washed in water and the reaction product was converted to lead sulphide. An
adjacent section was stained with haematoxylin and eosin.
Glycogen
Tissue sections were prepared similarly to those for immunofluorescence.
The sections were stained with periodic acid-SchifF (PAS). An adjacent section
was stained with haematoxylin and eosin.
To remove glycogen, digestion in diastase (Wako Pure Chemical Industries,
Ltd., Osaka) at a concentration of 0-1 % in PBS (pH 7-2) at 37 °C for 1 h was
performed. After digestion, sections were stained by the PAS technique.
142
N. SHIOJIRI
Table 1. AFP localization in the developing mouse liver
Stage
Hepatocytes
Intrahepatic
bile duct
cells
Hepatic
duct cells
Extrahepatic
bile duct
cells
Endothelial
cells
Haematopoietic
cells
8-5 g.d.
- ~+
90
- ~+
9.5
••—11-5
+++
13-5
++
14-5
++
15-5
+- +H
16-5
±~ + H
,
[
±~ + H
17-5
— ~±
Newborn
1—
—
±
1- +
*
Postnatal
+
*
*
+
*
7 days
Postnatal
+
*
*
+
*
14 days
Adult
*
*
*
t Presumptive hepatic endoderm.
t Precursor cells.
Under dotted lines, morphologically differentiated cells appear. - , no immunofluorescence;
± , weak immunofluorescence; + , moderate immunofluorescence; + + , strong immunofluorescence; + + + , very strong immunofluorescence.
* Not determined.
g.d., gestation days.
J^J
RESULTS
1. AFP localization
Developmental changes of the localization of AFP in the mouse liver are
summarized in Table 1.
Endodermal cells located in the anterior intestinal portal region became
positive for AFP in 8-5-day embryos (Fig. 1). At 9-0 days, the stratified endoderm of the hepatic diverticulum was weakly or strongly stained for AFP.
In 9-5-day foetuses, the cranial portion of the hepatic diverticulum was
strongly positive for AFP (Figs 2, 3). The staining intensity was similar between the cells on the luminal side and the hepatocytes of short sprouts in the
cranial diverticulum. In the endodermal cells of the caudal diverticulum, AFP
was absent or sometimes present on the luminal side.
In the 11 -5-day liver, all hepatocytes and endothelial cells were heavily stained
for AFP.
In the liver of 13-5-day foetuses, most hepatocytes were still brightly fluorescent, though the intensity became lower than that found in earlier stages. At
this stage in development, lumen structures appeared in the liver trabeculae.
Cytochemical differentiation of mouse liver cells
AIP
143
AIP
YS
1/1
1
-. -*
HD
2 il
3.1
Fig. 1. (A) Median sagittal section of the anterior intestinal portal (AIP) region of an
8-5-day embryo. Note an AFP-positive endodermal cell (arrow). YS, visceral yolk
sac. x 250. (B) Schematic design corresponding to Fig. 1 A.
Fig. 2. (A) Median sagittal section of a 9-5-day embryo. All of the endodermal cells
in the stratified cranial diverticulum show AFP immunofluorescence. HD, hepatic
diverticulum. x 250. (B) Schematic design corresponding to Fig. 2 A.
Fig. 3. (A) Hepatocytes of hepatic cords at 9-5 days. The cytoplasm is AFP-positive.
x 500. (B) Schematic design corresponding to Fig. 3 A.
2B
3B
Endodermal cells constituting the lumen structures were also stained for AFP
(Fig. 4). Epithelial cells of hepatic ducts connecting v/ith the lumen structures
showed strong intracellular labelling, particularly on their luminal side (Fig. 5).
Epithelial cells of extrahepatic bile duct were almost negative, except for
144
N. SHIOJIRI
r
\
• ••
PV
4B
PV
* • • > • . .
S3S
PV
5B
PV
Fig. 4. (A) AFP in endodermal cells of a luman structure (arrow) around the portal
vein (PV) at 13-5 days, x 500. (B) Schematic design corresponding to Fig. 4 A.
Fig. 5. (A) AFP immunofluorescence in the liver of a 13-5-day foetus. Hepatic duct
cells (arrow) and connective tissue around the extrahepatic bile duct (ED) are positive.
PV, portal vein, x 250. (B) Schematic design corresponding to Fig. 5 A.
Fig. 6. (A) AFP immunofluorescence in the liver of a 16-5-day foetus. Flat epithelial
cells (arrow) of a biliary space are negative. PV, portal vein, x 500. (B) Schematic
design corresponding to Fig. 6 A.
6B
Cytochemical differentiation of mouse liver cells
145
occasional staining on their luminal side, whereas the underlying connective tissue
was heavily labelled. Haematopoietic cells, present, in clusters, were negative.
AFP immunofluorescence in hepatocytes became weak in intensity at 15-5
days. Endodermal cells constituting the lumen structures were still stained for
AFP. At this stage, endothelial cells of the hepatic veins showed the strongest
staining intensity in the liver. Those of the portal veins were weakly stained.
At 16-5 and 17-5 days, hepatocytes were stained with a lower intensity, though
a small proportion of hepatocytes contained much AFP and was distributed
randomly in the liver parenchyma (Fig. 6). Most epithelial cells of the intrahepatic bile ducts were negative. A few intrahepatic bile duct cells were fluorescent on their luminal side.
In the liver of newborn mice, the endothelial cells were still heavily labelled
for AFP. Most hepatocytes contained little or no AFP. Some hepatocytes
located near veins or on the edges of hepatic lobes were still positive.
Tn the liver of 7-day-old mice, the pattern of AFP localization was similar
to that observed in the newborn liver. Non-fluorescent hepatocytes increased
in number. Epithelial cells of the intrahepatic bile ducts were not labelled.
At 14 days, a few hepatocytes near blood vessels and on the edges of hepatic
lobes were still AFP-positive.
The adult liver contained no detectable AFP. Control sections were
invariably negative.
2. Localization of ALP activity
Developmental changes in the localization of ALP activity in mouse livers are
summarized in Table 2.
ALP activity was seen in the endodermal cells of the anterior intestinal portal
region at 9-0 days. Cell membrane of the endodermal cells located on the luminal
side was positive. At 8-5 days, no ALP activity was seen in the endodermal cells
of the foregut.
In 9-5-day embryos, both the cranial and caudal portions of the hepatic
diverticulum showed strong ALP activity.
Liver parenchyma of 11-5-day foetuses was ALP-negative, though presumptive gall bladder epithelium was positive.
At 13-5 days, cell membrane of some hepatocytes showed strong ALP activity
again (Fig. 7). ALP-positive hepatocytes were distributed randomly in the liver
parenchyma. Endodermal cells of the lumen structures were negative. Epithelium
of the extrahepatic bile duct, except that of the presumptive gall bladder
region, was also negative.
With the progress of liver development, hepatocytes with ALP-positive cell
membrane increased in number. At 15-5 days, many hepatocytes possessed ALP
activity. All epithelial cells of the extrahepatic bile duct were ALP-negative.
In the liver of 16-5-day foetuses, epithelial cells of the intrahepatic bile ducts
were negative (Fig. 8).
146
N. SHIOJIRI
Table 2. Localization of ALP activity in the developing mouse liver
Stage
Hepatocytes
8-5 g.d.
90
9-5
11-5
13-5
14-5
15-5
16-5
17-5
Newborn
Postnatal
7 days
Postnatal
14 days
Adult
-X
Intrahepatic Extrahepatic
bile duct
bile duct
Endothelial
cells
cells f
cells
Haematopoietic
cells
+x
+
— +
—~+
+
++
++
- ~++
—
t Extrahepatic bile duct cells contain hepatic duct cells,
j Presumptive hepatic endoderm.
§ Precursor cells. Under dotted lines, morphologically differentiated cells appear,
no activity; ± , weak activity; + , moderate activity; + + , strong activity.
* Not determined,
g.d., gestation days.
ALP activity was present in hepatocytes of 7-day-old mice. In 14-day-old
and adult liver, no ALP activity was observed, though weak activity was seen
in unfixed frozen sections.
ALP activity in hepatocytes was always inhibited by the presence of 1 mM
L-cysteine. Sections incubated in substrate-free medium were negative.
3. Localization of 5'-Nase activity
The localization of 5'-Nase activity is summarized in Table 3.
In the liver of 14-5-day foetuses, weak 5'-Nase activity appeared in the cell
membrane of a small proportion of hepatocytes. The epithelium of the extrahepatic bile duct containing hepatic ducts was negative.
At 15-5 days, the hepatocytes with 5'-Nase activity increased in number.
In the liver of 16-5-day foetuses, the epithelial cells of the intrahepatic bile
ducts derived from the endodermal cells constituting the lumen structures were
negative.
In the liver of newborn mice, cell membrane of most hepatocytes was 5'-Nasepositive as that in the earlier stages. The strongest activity was seen in the cell
membrane of hepatocytes of adult mice. Some endothelial cells were also
positive.
Cytochemical differentiation of mouse liver cells
147
PV
Fig. 7. ALP activity in the liver of a 13-5-day foetus. Endodermal cells of a lumen
structure (arrow) show no activity. PV, portal vein, x 450.
Fig. 8. ALP activity in the liver of a 16-5-day foetus. Flat epithelial cells (arrow) of a
biliary space show no activity. PV, portal vein, x 450.
Non-specific ALP activity was not observed at pH 7-2. Sections incubated
with substrate-free medium were negative.
4. Initiation of glycogen storage
Results are shown in Table 4.
Glycogen appeared in the cytoplasm of a small proportion of hepatocytes
from 15-5-day foetuses (Fig. 9). The liver cells in the earlier stages were negative.
Hepatocytes containing glycogen were localized in the central area of the liver.
PAS-positive substance in the hepatocytes could be digested by diastase. Some
endothelial cells of the portal veins also contained glycogen. The endodermal
cells of the lumen structures were negative.
In the liver of 16-5- and 17-5-day foetuses, hepatocytes rich in glycogen increased in number. The flat intrahepatic bile duct cells and hepatic duct cells
contained no glycogen.
Histochemical characteristics of the hepatic cells derived from the hepatic
endoderm are summarized in Fig. 10.
DISCUSSION
1. Differentiation of the hepatic cells from presumptive hepatic endoderm
AFP is present only in hepatocytes of developing livers of mammals (Najak &
Mital, 1977; Carlsson & Ingvarsson, 1979). However, in the present investigation, a few endodermal cells containing AFP in their cytoplasm appeared in
theforegut endoderm in the anterior intestinal portal region at 8-5 days. At 9-0
days, hepatic endodermal cells produced AFP, but the AFP production occurred
asynchronously among them. At 9-5 days, all of the stratified endodermal cells
of the cranial diverticulum contained much AFP in their cytoplasm. These
148
N. SHIOJIRI
Table 3. Localization of 5'-Nase activity in the developing mouse liver
Stage
Hepatocytes
Intrahepatic Extrahepatic
bile duct
bile duct Endothelial
cells
cellsf
cells
8-5 g.d.
90
9-5
*
11-5
12-5
13-5
14-5
- ~ +
15-5
+
16-5
+
17-5
+
Newborn
+
—
—
Adult
++
*
t Extrahepatic bile duct cells contain hepatic duct cells.
Under dotted lines, morphologically differentiated cells appear,
activity; +, moderate activity; + +, strong activity.
* Not determined,
g.d., gestation days.
Haematopoietic
cells
, no activity; ± , weak
Table 4. Glycogen storage in the developing mouse liver
Stage
Hepatocytes
8-5 g.d.
90
9-5
11-5
-1
-t
*"-
Intrahepatic Extrahepatic
bile duct
bile duct
cells
cells t
13-5
-
•
14-5
15-5
16-5
17-5
Newborn
Postnatal
7 days
Postnatal
14 days
Adult
-
!
-~ ++ ;
+- ++
++
+
4-
:
Endothelial
cells
Haematopoietic
cells
-§
-§
- § ;
:
-
-
-~ +
-~ +
—
—
*
—
-
*
*
+
-
*
-
*
+
-
*
-
*
f Extrahepatic bile duct cells contain hepatic duct cells.
X Presumptive hepatic endoderm.
§ Precursor cells.
Under dotted lines, morphologically differentiated cells appear. - , no storage; ± , weak
storage; + , moderate storage; + + , strong storage.
* Not determined,
g.d., gestation days.
Cytochemical differentiation of mouse liver cells
149
V*
B
Fig. 9. (A) Glycogen in the liver of a 15-5-day foetus. Endodermal cells of a lumen
structure contain no glycogen. PV, portal vein. x450. (B) A section adjacent
to that of Fig. 9 A. PV, portal vein. Haematoxylin and eosin. x450.
results suggest that these AFP-positive hepatic endodermal cells can differentiate into AFP-positive hepatocytes, epithelial cells of the intrahepatic bile
ducts, and those of the hepatic ducts in late embryonal stages (Fig. 10). The
presumptive hepatic endoderm stratifies and then the hepatic cords are extended
from them (Shiojiri, 1979). Strong staining for AFP was observed at the statification of the hepatic endoderm. The stratification of the hepatic endoderm, therefore, seems to be prerequisite to the differentiation of AFP-positive hepatocytes.
The caudal portion of the hepatic diverticulum differs histologically from the
cranial portion at 9-5 days (Shiojiri, 1979). We observed that, at 9-5 days, the
endodermal cells of the caudal diverticulum were stained only on their luminal
side for AFP. AFP was localized mainly in the cranial diverticulum. These
facts suggest that the developmental fate of the caudal diverticulum is different
from that of the cranial diverticulum. Most epithelial cells of the extrahepatic
bile duct develop from the caudal diverticulum (Fig. 10).
2. Maturation of hepatocytes type I to hepatocytes type II
Morphologically, basophilic hepatocytes type I differentiate into larger, nonbasophilic hepatocytes type II at 16-5-17-5 days (Shiojiri, 1979). By 15-5 days,
hepatocytes contained much AFP, but after 16-5 days hepatocytes with less or
no AFP increased in number. Hepatocytes of adult mice contained no detectable AFP. Therefore, AFP is thought to be a feature of hepatocytes type I. The
decrease in number of AFP-positive hepatocytes during perinatal life is in
accordance with the decline of the serum concentration of AFP (Najak & Mital,
1977; Carlsson & Ingvarsson, 1979).
The present study revealed that enzymic differentiation in a population of
hepatocytes type I occurs asynchronously, and that enzymic differentiation in
hepatocytes precedes morphological changes. ALP and 5'-Nase activities
Caudal portion
AFP(- ~ +)
ALP(+)
glycogen(-)
Hepatocytes
type I
AFP(+)
ALP(- -»• +)
5'-Nase(--»-+)
glycogen(- -*• +)
Day 10-5
\
Hepatic
duct cells
AFP(+)
ALP(-)
5'-Nase(-)
glycogen(-)
AFP(+)
ALP(-)
glycogen(-)
Endodermal cells
of lumen structures
Day 13-5
AFP(-)
ALP(-)
5'-Nase(-)
glycogen(-)
Other extrahepatic
bile duct cells
Hepatic duct cells
AFP(-)
ALP(-)
5'-Nase(-)
glycogen(-)
Hepatocytes
type II
AFP(+ -> - )
ALP(+)
5'-Nase(+)
glycogen(+)
Intrahepatic
bile duct cells
AFP(-)
ALP(-)
5'-Nase(-)
glycogen(-)
Day 16-5—17 5
Fig. 10. Differentiation of the epithelium of the mouse hepatic diverticulum. A F P , alphafoetoprotein; ALP, alkaline phosphatase;
5'-Nase, 5'-nucleotidase; —, not stained; + , stained.
Hepatic
diverticulum
Cranial portion
AFP(+)
ALP(+)
glycogen(-)
Gestation day 9-5
90
(-1
S
25
Cytochemical differentiation of mouse liver cells
151
appeared in a few hepatocytes at 13-5 and 14-5 days, respectively. After 15-5 days
many hepatocytes possessed these enzyme activities. The expression of enzyme
activities does not occur at the same time during development: hepatocytes
type I express ALP activity first, and then 5'-Nase activity. Glucose-6-phosphatase activity was reported to appear asynchronously among hepatocytes of rat
foetuses (Leskes, Siekevitz & Palade, 1971). We found in the present investigation that glycogen begins to be stored in some hepatocytes type I at 15-5 days
and this initiation precedes the morphological differentiation of hepatocytes
type I. This result of the asynchronous initiation of glycogen storage in hepatocytes is consistent with that obtained by Peters, Kelly & Dembitzer (1963).
Heavy glycogen accumulation can be regarded as a characteristic of hepatocytes
type II.
It has been suggested that AFP and albumin can be produced by one clone
of liver cells. In the young foetus the clone of liver cells produces AFP predominantly, but in the adult exclusively albumin (Najak & Mital, 1977; Carlsson &
Ingvarsson, 1979). The present investigation revealed that hepatocytes type I
contain much AFP and progressively express the characteristics of hepatocytes
type II, suggesting that a clone of hepatocytes type I differentiates into hepatocytes type II (Fig. 10).
3. Differentiation of intrahepatic bile duct cells
The intrahepatic bile ducts originate from lumen structures appearing in the
liver trabeculae at 13-5 days. The endodermal cells constituting the lumen structures are morphologically similar to both the young hepatocytes and epithelial
cells of the hepatic ducts (Shiojiri, 1979). Wilson et al. (1963) stated that the
intrahepatic bile ducts originate from the tubules consisting of hepatocytes
which can be identified by electron microscopy. It remains to be ascertained
(enzymo- and immunocytochemically) whether the precursor cells of the intrahepatic bile duct cells are hepatocytes or not. In the present investigation, the
endodermal cells of the lumen structures were found to be positive for AFP,
and negative for ALP and glycogen, and to be similar to hepatocytes type I.
Since differentiation of a small proportion of hepatocytes type I to hepatocytes
type II progressed when lumen structures were identified in the liver trabeculae,
the origin of the endodermal cells of these lumen structures was not from the
proportion of hepatocytes type I which differentiated into hepatocytes type II,
However, it is also possible that the cells of the lumen structures may be de
rived from the epithelial cells of the hepatic ducts, which they resemble structur
ally and which contain much AFP (particularly on their luminal side), no
glycogen, and no ALP and 5'-Nase activities. The flat epithelial cells of the
intrahepatic bile ducts show no AFP immunofluorescence, no ALP and 5'-Nase
activities, and no glycogen.
The results of the present cytochemical studies also imply that the endodermal
cells of the lumen structures are the intermediate cells between hepatocytes type
152
N. SHIOJIRI
I and epithelial cells of the hepatic ducts (Fig. 10). To clarify the precise
origin of the cells constituting the lumen structures is a problem for the future.
The author wishes to express his deep gratitude to Professor Takeo Mizuno of the University
of Tokyo for his invaluable advice and encouragement during the course of this work.
REFERENCES
W. (1926). The embryogenesis of human bile capillaries and ducts. Am. J. Anat. 36,
451-465.
CARLSSON, R. N. K. & INGVARSSON, B. I. (1979). Localization of a-fetoprotein and albumin
in pig liver during fetal and neonatal development. Devi Biol. 73, 1-10.
CHEDID, A. & NAIR, V. (1974). Ontogenesis of cytoplasmic organelles in rat hepatocytes and
the effects of prenatal phenobarbital on endoplasmic reticulum development. Devi Biol. 39,
49-62.
DE WOLF-PEETERS, C, DE VOS, R. & DESMET, V. (1972). Electron microscopy and histochemistry of canalicular differentiation in fetal and neonatal rat liver. Tissue & Cell 4,
379-388.
ENGELHARDT, N. V., GOUSSEV, A. I., SHIPOVA, L. JA. & ABELEV, G.I. (1971). Immunofiuorescent study of alpha-foeto protein (afp) in liver and liver tumors. I. Technique of
afp localization in tissue sections. Int. J. Cancer 7, 198-206.
ENZAN, H., OHKITA, T., FUJITA, H. & IIJIMA, S. (1974). Light and electron microscopic
studies on the development of periportal bile ducts of the human embryo. Ada Path. Jap.
24, 427-447.
GOMORI, G. (1939). Microtechnical demonstration of phosphatase in tissue sections. Proc.
Soc. exp. Biol. Med. 42, 23-26.
GREENGARD, O. (1969). Enzymic differentiation in mammalian liver. Science 163, 891-895.
GREENGARD, O., FEDERMAN, M. & KNOX, W. E. (1972). Cytomorphometry of developing rat
liver and its application to enzymic differentiation. J. Cell Biol. 52, 261-272.
HAMMER, J. A. (1926). Uber die erste Entstehung der nicht kapillaren intrahepatischen
Gallengange beim Menschen. Z. mikrosk.-anat. Forsch. 5, 59-89.
KOGA, A. (1971). Morphogenesis of intrahepatic bile ducts of the human fetus. Light and
electron microscopic study. Z. Anat. EntwickGesch. 135, 156-184.
LESKES, A., SIEKEVITZ, P. & PALADE, G. E. (1971). Differentiation of endoplasmic reticulum
in hepatocytes. I. Glucose-6-phosphatase distribution in situ. J. Cell Biol. 49, 264-287.
NAJAK, N. C. & MITAL, I. (1977). The dynamics of a-fetoprotein and albumin synthesis in
human and rat liver during normal ontogeny. Am. J. Path. 86, 359-374.
PETERS, V. B., KELLY, G. W. & DEMBITZER, H. M. (1963). Cytologic changes in fetal and
neonatal hepatic cells of the mouse. Ann. N.Y. Acad. Sci. Ill, 87-103.
PURTILO, D. T. & YUNIS, E. J. (1971). a-Fetoprotein: Its immunofluorescent localization in
human fetal liver and hepatoma. Lab. Invest. 25, 291-294.
SEVERN, C. B. (1972). A morphological study of the development of the human liver. II
Establishment of liver parenchyma, extrahepatic ducts and associated venous channels.
Am. J. Anat. 133, 85-108.
SHIOJIRI, N. (1979). The differentiation of the hepatocytes and the intra- and extrahepatic
bile duct cells in mouse embryos. /. Fac. Sci., Univ. Tokyo iv 14, 241-250.
TURCHINI, J. P. (1961). Le foie de Souris a la periode neonatale. Caracteres cytochimiques.
C. r. Seam. Soc. Biol. 155, 1502-1505.
WACHSTEIN, M. (1959). Enzymatic histochemistry of the liver. Gastroenterol. 37, 525-537.
WACHSTEIN, M. & MEISEL, E. (1957). Histochemistry of hepatic phosphatases at a physiologic
pH; with special reference to the demonstration of bile canaliculi. Am. J. Clin. Path. 27,
13-22.
WILSON, J. W., GROAT, C. S. & LEDUC, E. H. (1963). Histogenesis of the liver. Ann. N.Y.
Acad. Sci. Ill, 8-24.
BLOOM,
(Received 30 April 1980, revised 30 September 1980)