Differentiation of the endodermal cells of the mouse liver was studied enzymo- and immunocytochemically by analyzing the cellular localization of alphafoeto protein (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. AFP-containing 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.

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 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 al. 1963; Enzan, Ohkita, Fujita & lijima, 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.

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.

Immunofluorescent 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 μm 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) 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 FITC-conjugated 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 μm 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’-Nose activity

Tissue specimens for 5′-Nase activity were frozen in hexane cooled by dry ice-ethanol. Frozen sections were cut at 6 μm 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 β-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.

1. AFP localization

Developmental changes of the localization of AFP in the mouse liver are summarized in Table 1.

Table 1

AFP localization in the developing mouse liver

AFP localization in the developing mouse liver
AFP localization in the developing mouse liver

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.

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.·250. (B) Schematic design corresponding to Fig. 1 A.

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.·250. (B) Schematic design corresponding to Fig. 1 A.

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.

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, × 250. (B) Schematic design corresponding to Fig. 2A.

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, × 250. (B) Schematic design corresponding to Fig. 2A.

Fig. 3

(A) Hepatocytes of hepatic cords at 9·5 days. The cytoplasm is AFP-positive. × 500. (B) Schematic design corresponding to Fig. 3 A.

Fig. 3

(A) Hepatocytes of hepatic cords at 9·5 days. The cytoplasm is AFP-positive. × 500. (B) Schematic design corresponding to Fig. 3 A.

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 fluor-escent, though the intensity became lower than that found in earlier stages. At this stage in development, lumen structures appeared in the liver trabeculae.

Endodermal cells constituting the lumen structures were also stained for AFP (Fig. 4). Epithelial cells of hepatic ducts connecting with 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 occasional staining on their luminal side, whereas the underlying connective tissue was heavily labelled. Haematopoietic cells, present in clusters, were negative.

Fig. 4

(A) AFP in endodermal cells of a luman structure (arrow) around the portal vein (PV) at 13·5 days, × 500. (B) Schematic design corresponding to Fig. 4A.

Fig. 4

(A) AFP in endodermal cells of a luman structure (arrow) around the portal vein (PV) at 13·5 days, × 500. (B) Schematic design corresponding to Fig. 4A.

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. ×250. (B) Schematic design corresponding to Fig. 5 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. ×250. (B) Schematic design corresponding to Fig. 5 A.

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 intra-hepatic bile ducts were negative. A few intrahepatic bile duct cells were fluorescent on their luminal side.

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, × 500. (B) Schematic design corresponding to Fig. 6 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, × 500. (B) Schematic design corresponding to Fig. 6 A.

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.

In 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.

Table 2

Localization of ALP activity in the developing mouse liver

Localization of ALP activity in the developing mouse liver
Localization of ALP activity in the developing mouse liver

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.

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, × 450.

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, × 450.

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).

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, × 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, × 450.

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.

Table 3

Localization of 5′-Nase activity in the developing mouse liver

Localization of 5′-Nase activity in the developing mouse liver
Localization of 5′-Nase activity in the developing mouse liver

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 extra-hepatic 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′-Nase-positive 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.

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.

Table 4

Glycogen storage in the developing mouse liver

Glycogen storage in the developing mouse liver
Glycogen storage in the developing mouse liver

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.

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. ×450. (B) A section adjacent to that of Fig. 9 A. PV, portal vein. Haematoxylin and eosin. ×450.

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. ×450. (B) A section adjacent to that of Fig. 9 A. PV, portal vein. Haematoxylin and eosin. ×450.

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.

Fig. 10

Differentiation of the epithelium of the mouse hepatic diverticulum. AFP, alphafoetoprotein; ALP, alkaline phosphatase; 5′-Nase, 5′-nucleotidase; –, not stained; +, stained.

Fig. 10

Differentiation of the epithelium of the mouse hepatic diverticulum. AFP, alphafoetoprotein; ALP, alkaline phosphatase; 5′-Nase, 5′-nucleotidase; –, not stained; +, stained.

1. Differentiation of the hepatic cells from presumptive hepatic endoderm

AFP is present only in hepatocytes of developing livers of mammals (Najak & Mitai, 1977; Carlsson & Ingvarsson, 1979). However, in the present investigation, a few endodermal cells containing AFP in their cytoplasm appeared in the foregut 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 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, non-basophilic 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 & Mitai, 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 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 & Mitai, 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 fiat 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 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.

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