Immunolocalization of extracellular matrix components and integrins during mouse liver development



Intrahepatic biliary cell differentiation takes place in periportal hepatoblasts under the influence of the subjacent connective tissue, the mechanism of which is still unclear. This study was undertaken to analyze the immunolocalization of extracellular matrix components and their cellular receptors during mouse liver development, with special attention given to biliary differentiation and vascular development. In young fetal mouse liver, primitive structures of sinusoids were developed between hepatic cords associated with hematopoietic cells demonstrated by immunohistochemistry of basal laminar components, the α6 integrin subunit, and PECAM-1. Portal veins and hepatic veins showed different staining intensities of α2, α3, and α6 integrin subunits from early stages of development. Anti-β4 integrin subunit antibodies reacted with portal veins, but not with hepatic veins after perinatal stages. Their different phenotypes may be related to the preferential differentiation of periportal bile ducts. In intrahepatic bile duct development, periportal hepatoblasts adjacent to the connective tissue were immunostained for each basal laminar component on the basal side at almost the same time; α3, α5, α6, and β4 integrin subunits were immunohistochemically detectable later than the basal laminar components. These staining patterns of intrahepatic bile duct cells clearly differed from those of extrahepatic bile duct cells from the beginning of their development, suggesting that these ducts are of different origins. In conclusion, the vascular structures, including sinusoids, portal veins, and hepatic veins, develop from early stages of liver development, and the extracellular matrix components may play important roles in biliary differentiation and vascular development. Supplementary material for this article can be found on the HEPATOLOGY website ( (HEPATOLOGY 2004;40:346–355.)

Mature hepatocytes and intrahepatic biliary epithelial cells share the common origin of hepatoblasts, which are immature hepatocytes in fetal stages and express α-fetoprotein and albumin but no mature hepatocyte markers such as urea-cycle enzymes.1, 2 Periportal connective tissue induces the subjacent hepatoblasts to differentiate into biliary epithelial cells,3 and during their differentiation, laminin, peanut agglutinin (PNA)-binding sites and bile duct-type cytokeratins are expressed.4–9 Recent studies have shown that Jagged1 and Notch2 expressed in periportal cells are involved in that differentiation.10 However, their differentiation process is not fully understood. It remains to be studied what is the sequence by which basal laminar components such as type IV collagen, laminin, nidogen, heparan sulfate proteoglycan (HSPG), and PNA-binding sites and their cellular receptors (called integrins) appear in biliary cell differentiation. Such markers can also precisely characterize biliary cell differentiation from hepatoblasts cultured in vitro. It is well known that intrahepatic bile ducts differentiate around portal veins, but not in other hepatic regions, which include pericentral and midzonal regions.11–13 Portal veins, hepatic veins (central veins), and sinusoids develop from the omphalomesenteric veins, but sinusoids are also possibly derived from the septum transversum mesenchyme.14, 15 Investigation of the mechanisms by which these three types of blood vessels differentiate during liver development may contribute to an understanding of preferential development of periportal bile ducts. The development of the hepatic artery, which is located in the portal area, is disturbed by the gene inactivation of HNF6 and HNF1β, both of which play important roles in biliary cell differentiation.16, 17

Studies with cytokeratin antibodies and some fluorescent lectins demonstrated the different reactivities of the extrahepatic and intrahepatic bile ducts in their early development,4–6, 18 suggesting the dual origin of the whole biliary duct system. The progenitor cells of both ducts transiently express α-fetoprotein.1 Further immunohistochemical characterization of the extrahepatic bile duct development may provide insights into not only its developmental origin but also into the establishment of the whole biliary duct system.

The differentiation and growth of hepatoblasts from the hepatic endoderm and maturation of fetal hepatocytes may depend on their interactions with the surrounding mesenchyme, including extracellular matrices (ECMs) or blood vessels.19–23 Although there are a few studies on the localization of ECM components and blood vessel formation in liver development,24–26 the positional relationships among the ECM components, or among hepatoblasts (hepatocytes), blood vessels, and ECM components are poorly understood. Extensive screening of ECM components and the integrins expressed—as well as vascular system differentiation during liver development—would also be useful in understanding of the mechanisms of hepatocyte maturation.

In the present study, we analyzed immunolocalization of ECM components and their cellular receptors throughout mouse liver development with attention given to the development of the biliary duct system and blood vessels as well as hepatocyte maturation. We report phenotypic differences of portal and hepatic veins in early stages, the development of hepatic sinusoids from early fetal to postnatal stages, and differentiation processes of the biliary duct system.


PNA, peanut agglutinin; HSPG, heparan sulfate proteoglycan; ECM, extracellular matrix; WGA, wheat germ agglutinin.

Materials and Methods


C3H/HeSlc strain mice (SLC, Shizuoka, Japan) were used. Animals were mated at night, and noon of the day the vaginal plug was found was considered 0.5 days' gestation. Fetuses at 9.5–17.5 days' gestation, newborn (1 day old), 1-week-old and 2-week-old young, and adult animals (8 weeks old) were used. At least three animals and five sections for each animal were examined in immunohistochemistry.


Tissues were frozen in n-hexane cooled with dry ice-ethanol. Frozen sections were cut into 8-μm slices and fixed in cold acetone (−30°C) for 10 minutes. Paraffin sections were also examined for very young fetuses at 9.5, 10.5, and 11.5 days' gestation. Whole fetuses were fixed in a chilled mixture of glacial acetic acid and 95% ethanol (99:1 v/v) overnight, and embedded in paraffin. Paraffin sections were cut 6-μm thick.

Frozen sections or dewaxed sections were incubated with primary antibodies for 1 hour at room temperature (Table 1). After being thoroughly washed with phosphate-buffered saline, sections were incubated with fluorescein- or Cy3-labeled donkey anti-rabbit, -rat, or -guinea pig immunoglobulin G antibodies (1/50 dilution for fluorescein-labeled antibodies and 1/500 dilution for Cy3-labeled antibodies; Jackson ImmunoResearch Laboratory, West Grove, PA) for 1 hour at room temperature. The specific immunofluorescence was observed with a fluorescence microscope (model BX50 equipped with BX-FLA; Olympus, Tokyo, Japan). Double immunostaining was also performed between the antigens whose antisera were raised in different species. In negative control sections, the incubation with the primary antibodies was omitted.

Table 1. Primary Antibodies Used in Immunohistochemistry
rabbit anti-rat type I collagen antiserumChemicon International, Temecula, CA1:100
rabbit anti-rat type III collagen antiserumChemicon International, Temecula, CA1:100
rabbit anti-mouse type IV collagen antiserumChemicon International, Temecula, CA1:100
rabbit anti-human fibronectin antiserumOrganon Teknica, West Chester, PA1:200
rabbit anti-mouse laminin antiserumE-Y Lab., San Mateo, CA1:200
rat monoclonal anti-mouse nidogen antibodyChemicon International, Temecula, CA1:200
rat monoclonal anti-HSPG antibodyChemicon International, Temecula, CA1:200
rabbit anti-integrin α1 antiserumChemicon International, Temecula, CA1:100
rabbit anti-integrin α2 antiserumChemicon International, Temecula, CA1:100
rabbit anti-integrin α3 antiserumChemicon International, Temecula, CA1:100
rabbit anti-integrin α5 antiserumChemicon International, Temecula, CA1:100
rat monoclonal anti-integrin α6 antibodyChemicon International, Temecula, CA1:100
rat monoclonal anti-integrin β1 antibodyChemicon International, Temecula, CA1:100
rat monoclonal anti-integrin β4 antibodyBD Biosciences, Tokyo, Japan1:100
rat monoclonal anti-PECAM-1 antibodyeBioscience, San Diego, CA1:100
rabbit anti-calf keratins antiserumDako, Carpinteria, CA1:300
guinea pig anti-bovine cytokeratin 8 and 18 antiserumProgen Biotechnik Gmbh, Heidelberg, Germany1:200

Dewaxed sections were also stained for fluorescein-labeled PNA or wheat germ agglutinin (WGA) (50 and 3 μg/mL, respectively; Vector Laboratories, Burlingame, CA).7


Adult Liver.

In adult mouse liver, laminin, nidogen, type IV collagen and HSPG were distributed in portal veins, central veins and sinusoids, and on the basal side of biliary epithelial cells and under the mesothelium (Supplemental Fig. 1C and 1E–G). Positive immunoreaction of laminin and nidogen around sinusoids was faint. Connective tissues of portal veins were strongly stained for nidogen and laminin. The staining of fluorescein-labeled WGA resembled that of HSPG. Types I and III collagen were abundantly distributed in connective tissue of portal and central veins, while some fibrous immunostaining in the perisinusoidal space was visible but not abundant (Supplemental Fig. 1A and 1B). Portal and central veins were strongly immunostained for fibronectin, but its immunostaining in sinusoids was comparatively weak (Supplemental Fig. 1D). In paraffin sections of adult mouse liver, PNA-binding sites were detected only in biliary epithelial cells.

Endothelial cells of blood vessels—including portal veins, central veins, and sinusoids—were positive for α1, α2, α3, α5, α6, and β1 subunits of integrin and PECAM-1, although their staining intensities varied among blood vessels and staining intensities in portal veins were generally stronger than those of central veins and sinusoids (Supplemental Fig. 2A–E and 2H). The endothelial staining in sinusoids and central veins for α3 and α6 integrin subunits was faint, and that of sinusoids was weak for PECAM-1. Anti-α3 integrin subunit antiserum also stained periportal connective tissue. β4 Integrin subunits were expressed in most endothelial cells of portal veins but not in those of central veins (Supplemental Fig. 2F and 2G). Intrahepatic biliary epithelial cells clearly expressed α3, α5, α6, and β4 subunits of integrin (Supplemental Fig. 2B–D and 2G).

Early Liver Development.

At 9.5 days' gestation, the liver primordium develops as a diverticulum in the ventral foregut in the anterior intestinal portal region. Although the basal lamina in the endodermal cells of the liver primordium was reactive with anti-laminin, nidogen, type IV collagen, fibronectin, and HSPG antibodies, the sprouts of the hepatic cords, which invaded the subjacent septum transversum mesenchyme, were not reactive or only very weakly stained (Fig. 1A–F). The subjacent mesenchyme was immunostained for fibronectin and HSPG. The proximal parts of the hepatic cords were strongly positive, but distal parts of hepatic cords were weakly or granularly immunostained by antisera against the basal laminar components in 10.5-day liver primordium (Fig. 1G–I). Fibronectin was also distributed around blood vessels and hepatoblasts. Types I and III collagen were not immunohistochemically detectable at these stages. Cells of hepatic cords were negative for α6 integrin subunits, whereas other foregut endodermal cells strongly expressed this subunit of integrin (Fig. 2A and 2B). The antibody also stained endothelial structures in the septum transversum mesenchyme and the omphalomesenteric veins and visualized the branching of the latter without the invasion of hepatic cords (see Fig. 2A and 2B). α6 Integrin subunit-positive sinusoidal structures were developed subjacent to the hepatic diverticulum. Anti-β1 integrin subunit antiserum was very weakly reactive with the cells of the hepatic diverticulum, but moderately with the foregut endodermal cells (Fig. 2C and 2D). This antiserum also stained the septum transversum mesenchyme and the blood vessels. No clear immunostaining for the β4 subunit of integrin was detected in 9.5- to 10.5-day embryos, including in the liver primordium.

Figure 1.

Expression of ECM components in early liver development: (A–F) 9.5-day liver primordium and (G–I) 10.5-day liver primordium. (A, G) Hematoxylin-eosin staining. (B, H) Fibronectin immunostaining. (C) Type IV collagen immunostaining. (D) Laminin immunostaining. (E, I) Nidogen immunostaining. (F) HSPG immunostaining. All basal laminar components are weakly immunostained in some parts of the 9.5-day hepatic diverticulum, where hepatic cords start to invade the septum transversum mesenchyme (arrowheads, B–F). In 10.5-day liver primordium, the hepatic cords are delineated by linear or granular staining of the basal laminar components (H, I). Nidogen immunostaining in distal parts of hepatic cords tends to be granular. Arrows indicate basal laminar component-positive or -negative hepatic cords (H, I). Vascular development is abundant in 10.5-day livers (asterisks, G–I). Bar = 50 μm. FG, foregut; HD, hepatic diverticulum; ST, septum transversum mesenchyme; HSPG, heparan sulfate proteoglycan.

Figure 2.

Expression of (A, B) α6 and (C, D) β1 integrin subunits in 9.5-day liver primordium. Foregut endodermal cells express α6 and β1 integrin subunits, whereas cells of the hepatic diverticulum are very weakly positive. Endothelial cells differentiate close to the hepatic diverticulum (arrow, B). Arrowheads indicate branching of the omphalomesenteric vein without the invasion of hepatic cords (B). Bars = 50 μm. NT, neural tube; FG, foregut; HD, hepatic diverticulum; OV, omphalomesenteric vein; ST, septum transversum mesenchyme.

Vascular Development, Hepatoblasts, and Hepatocytes.

In 11.5- or 12.5-day livers, the primitive structures of the sinusoids were already recognizable between hepatic cords as observed in 13.5- and 14.5-day livers, which were associated with hematopoietic cells, by immunohistochemistry of ECM components, the α6 subunit of integrin, and PECAM-1 (Fig. 3A–H). The α6 integrin subunit and PECAM-1–positive cells, on which the basal laminar components were deposited, formed narrow lumina (see Fig. 3A–H; Fig. 4A–C). Laminin and nidogen immunoreactivities in these structures were weak and granular in sections (Supplemental Fig. 3A, 3D, and 3G). The granular basal laminar components were also deposited close to hepatoblasts (Supplemental Fig. 3A–C). Not all cells expressing the α6 integrin subunit were positive for PECAM-1 immunostaining, and α6 integrin subunit-positive cells were neither hepatoblasts nor desmin-positive stellate cells (Fig. 4D–I). Desmin-positive stellate cell precursors surrounded the primitive sinusoidal structures but did not always colocalize with the basal laminar components (Supplemental Fig. 3D–F). The basal laminar components, including laminin, nidogen, HSPG and type IV collagen exhibited a similar distribution in the sinusoidal structures, portal veins, and hepatic veins (Supplemental Fig. 3G–O). Type IV collagen and HSPG showed broader distribution than laminin and nidogen, which almost perfectly colocalized in double immunofluorescence (see Supplemental Fig. 3J–O). Portal veins and hepatic veins showed stronger or continuous staining for the basal laminar components and were different from the sinusoidal structures, though the immunoreactivity of portal veins to the basal laminar components was generally stronger than that of hepatic veins in fetal livers.

Figure 3.

Primitive sinusoidal structures in (A) 10.5-day, (D, F–H) 12.5-day, (B) 13.5-day, (E) 14.5-day, and (C) 15.5-day livers. (A–C) α6 Integrin subunit immunostaining. (D, E) PECAM-1 immunostaining. (F) Hematoxylin-eosin staining. (G) Nidogen immunostaining. (H) Fibronectin immunostaining. α6 Integrin subunit or PECAM-1-positive vascularized structures are well developed from the early stages (arrows, A–E). Vascular development is obvious in 12.5-day livers (arrows, F), and fibronectin is present around vascular spaces (large arrowheads, H). Asterisk indicates weak and granular nidogen staining of vascular structures (G). The extrahepatic bile duct is clearly positive for nidogen on its basal side (G). Small arrowheads indicate prospective hepatic duct regions (F, G), which are lined with nidogen immunostaining (G). Bars = 50 μm. ED, extrahepatic bile duct; PV, portal vein; HV, hepatic vein; V, large vein.

Figure 4.

Double immunofluorescent analysis of (A, D, G) α6 integrin subunit and (B) laminin, (E) cytokeratins, or (H) desmin localization in 14.5-day fetal mouse liver. (C, F, I) Double exposure of A and B, D and E, and G and H, respectively. (A–C) Laminin (asterisks) is immunolocalized close to α6 integrin subunit-positive cells (arrowheads). (D–F) α6 Integrin subunit signals (arrowheads) do not overlap with cytokeratin immunostaining, which shows hepatoblasts (asterisks). (G–I) Desmin-positive hepatic stellate cells (asterisks) are immunolocalized close to α6 integrin subunit-positive cells (arrowheads). Bar = 50 μm. HV, hepatic vein; PV, portal vein.

Immunostaining in the liver parenchyma or in sinusoidal structures for the basal laminar components distributed comparatively evenly in midgestation stages (Fig. 5F and 5G), but those of laminin and nidogen became much stronger in periportal areas by 17.5 days (Fig. 5N and 5O). Periportal connective tissue was immunostained for laminin, nidogen, type IV collagen, and HSPG, while the staining in pericentral connective tissues was very weak. The distribution of the basal laminar components in sinusoidal structures became strong and continuous with development (Fig. 5D, 5H, 5L, and 5P), and adult-type distribution was established in 2-week-old livers. Fluorescent WGA stained primitive vascular spaces in fetal livers and stained sinusoids clearly and continuously in postnatal liver development (Supplemental Fig. 4A and 4B).

Figure 5.

Expression of ECM components during sinusoid and intrahepatic bile duct development. (A–H) 14.5-day liver. (I–P) 17.5-day liver. (A, I) Hematoxylin-eosin staining. (B, J) Type I collagen immunostaining. (C, K) Type III collagen immunostaining. (D, L) Type IV collagen immunostaining. (E, M) Fibronectin immunostaining. (F, N) Laminin immunostaining. (G, O) Nidogen immunostaining. (H, P) HSPG immunostaining. Arrows indicate epithelial cells of intrahepatic bile ducts or their precursors. Types I and III collagen (asterisks) are localized to the portal and hepatic veins, but also to liver parenchymal regions in lesser amounts (B, C, J, K). Type IV collagen, laminin, nidogen, and HSPG are immunostained in biliary epithelial cells and their precursors (D, G, H, L, N, O). Note stronger immunostaining of periportal areas for laminin and nidogen in 17.5-day livers (N, O). Sinusoidal structures are moderately positive for type IV collagen, fibronectin, and HSPG over both 14.5- and 17.5-day liver parenchymal regions (small arrowheads, D, E, H, L, M, P). Large arrowhead indicates hepatic ducts that have a lumen and are positive for laminin (F). Bars = 50 μm. PV, portal vein; ED, extrahepatic bile duct; HV, hepatic vein; HSPG, heparan sulfate proteoglycan.

Type I and III collagen signals were not detectable in young liver primordia, such as 10.5- and 11.5-day liver. From 14.5 days' gestation on, their signals became stronger. Such signals were localized in the primitive sinusoidal structures, that is, between the hepatic cords accompanied by hematopoietic cells (Fig. 5A–C). Portal veins and hepatic veins showed strong immunoreactivity for these collagens. Periportal connective tissue was more reactive. Their staining intensity became strong with development (Fig. 5I–K). Types I and III collagen were immunolocalized near basal laminar components, but strict colocalization was not seen (Supplemental Fig. 3G–I).

In young fetal mouse liver, portal veins and central veins also showed different staining intensities of α2, α3, and α6 subunits of integrin: namely, those in portal veins were generally stronger. Cells of portal veins were positively stained for the α2 subunit of integrin, but those of hepatic veins were negative (Fig. 6A and 6C). Periportal connective tissues were clearly stained for the α3 subunit of integrin after 17.5 days' gestation (Fig. 6D). The α6 subunit immunostaining in sinusoidal structures decreased after 17.5 days (i.e., the neonatal stage) (Fig. 6G). β1 Subunit expression was seen in blood vessels and their connective tissues and increased in sinusoidal structures after 17.5 days (Fig. 6B and 6H). α1 Integrin subunits were also immunolocalized in sinusoids after the neonatal period (Fig. 6F). β4 Integrin subunit expression also started in endothelial cells of portal veins in 17.5-day livers (Fig. 6E and 6I). Endothelial cells of hepatic veins were negative for the β4 subunit. PECAM-1 expression was detected in blood vessels throughout development, though it decreased in sinusoidal structures after the neonatal stage.

Figure 6.

Expression of (F) α1, (A, C) α2, (D) α3, (G) α6, (B, H) β1, and (E, I) β4 integrin subunits during hepatic vascular development. (A, B) 14.5-day liver. (C–E) 17.5-day liver. (F–I) Neonatal liver. α2 Integrin subunit expression is different between portal veins and hepatic veins; cells of portal veins express this integrin subunit more strongly (A, C). Periportal connective tissues express the α3 integrin subunit (D). Endothelial cells of portal veins express β4 integrin subunits (arrowheads, E, I). The expression of α1 and β1 integrin subunits in sinusoidal cells of neonatal liver is moderate (F, H), though the β1 integrin subunit expression is comparatively weak in 14.5-day liver (B). Arrows indicate α3, α6, and β4 integrin subunit expression in periportal bile duct structures in 17.5-day and neonatal livers (D, E, G, I). Free cells are α2 integrin subunit-positive (asterisk, C). Bar = 50 μm. HV, hepatic vein; PV, portal vein.

Although basal laminar components and fibronectin were deposited around hepatoblasts and hepatocytes during development (Fig. 5E and 5M for fibronectin), they showed no clear integrin subunit expression among the subunits examined compared with biliary epithelial cells.

Intrahepatic Bile Duct Development.

Tables 2 and 3 summarize the immunolocalization of ECM components and expression of integrins during mouse intrahepatic bile duct development, respectively.

Table 2. Immunolocalization of ECM Components in Mouse Intrahepatic Bile Duct Development
 13.5 g.d.14.515.517.5NB1-week-old2-week-oldadult
  1. +, positive immunostaining; −, negative immunostaining; g.d., gestation days; NB, newborn.

type IV collagen++++++++
PNA-binding sites−∼+−∼++++++
Table 3. Expression of Integrin Subunits in Mouse Intrahepatic Bile Duct Development
integrin subtype13.5 g.d.14.515.517.5NB1-week-old2-week-oldadult
  1. +, positive immunostaining; −, negative immunostaining; g.d., gestation days; NB, newborn.


At 13.5 and 14.5 days' gestation, the liver parenchyma in the pericentral and midozonal areas was moderately immunostained for laminin and nidogen, but by 17.5 days, that immunostaining was concentrated in the periportal area (portal veins and biliary cells) (see Fig. 5F, 5G, 5N, and 5O). Intrahepatic biliary duct development started in the liver parenchyma at 13.5–14.5 days' gestation with lumen opening and basal laminar formation (stronger expression of laminin, nidogen, type IV collagen, and HSPG) in periportal hepatoblasts (see Fig. 5A, 5D, 5F, 5G, and 5H and Supplemental Fig. 3A–C and M–O), but expression of biliary cell-type integrins appeared a little later. At 14.5–15.5 days' gestation, the basal surfaces were very weakly or moderately immunostained for α3, α5, and α6 subunits (Fig. 7A–C), and at 17.5 days this staining became clearer (Fig. 7D and 7E). PNA-binding sites appeared on the basal surfaces of periportal hepatoblasts at 14.5 days' gestation (Fig. 7F). The β4 subunit of integrin started to be expressed in some periportal biliary progenitors in 15.5-day livers. In neonatal livers, epithelial cells of intrahepatic bile ducts strongly expressed α3, α5, α6, and β4 subunits (see Fig. 6G and 6I).

Figure 7.

Expression of(A) α3, (B, D) α5, and (C, E) α6 integrin subunits and (F) PNA-binding sites during intrahepatic bile duct development. (A–C, F) 14.5-day liver. (D, E) 17.5-day liver. Periportal connective tissue starts to express the α3 integrin subunit (arrowhead, A). Free cells are α3 integrin subunit-positive (asterisk, A). Expression of the α5 integrin subunit is not seen in 14.5-day liver (B). α6 Integrin subunit expression is clearly seen in periportal biliary structures (arrow, C). The expression of α5 and α6 integrin subunits in periportal bile duct structures is obvious or abundant in 17.5-day livers (arrows, D, E). In the case of α6 integrin subunit staining, periportal connective tissue is also strongly positive (C, E). The basal surface of bile duct structures is PNA-positive (arrow, F). Bars = 50 μm. PV, portal vein; PNA, peanut agglutinin.

Extrahepatic Bile Duct Development.

The primordium of the gall bladder and extrahepatic bile ducts developed close to the liver primordium at 9.5–10.5 days' gestation. They consisted of a pseudostratified or simple columnar epithelium lined with the basal laminar components on the basal side (Supplemental Fig. 5A) and expressed α3, α5, and α6 subunits of integrin, especially on the basal side, throughout fetal liver development (Supplemental Fig. 5B and 5C). Hepatic ducts connecting the liver parenchyma with the extrahepatic bile duct started to be formed at 12.5 or 13.5 days' gestation, and their epithelial cells, which took the histology of a cell mass of hepatoblasts, were lined with basal laminar components but did not express α3, α5, and α6 subunits of integrin (see Fig. 3F and 3G and Supplemental Fig. 5B and 5C). After 14.5 days' gestation, hepatic ducts had lumina and were lined with low columnar epithelial cells on laminin-, nidogen-, type IV collagen-, and HSPG-positive basal lamina (see Fig. 5F). These cells gradually expressed α3, α5, and α6 subunits of integrin with development.

Other Cells.

Nonparenchymal cells—including megakaryocytes and some hematopoietic cells—in fetal mouse livers also expressed α2, α6, and β1 integrin subunits (see Fig. 6C). The α3 subunit was also expressed in some hematopoietic cells of 13.5- to 15.5-day livers (see Fig. 7A and Supplemental Fig. 5B). Hepatic capsular cells exhibited strong immunoreactivity to nidogen, laminin, HSPG, and type IV collagen in fetal livers. They were also positively immunostained for α3 and α5 integrin subunits.


The present study demonstrated that immunolocalization of ECM components and their cellular receptors changed with liver development, being correlated with the liver primordium formation, the development of the hepatic vascular system and biliary system, and hepatocyte maturation (Fig. 8).

Figure 8.

Expression of ECM components and integrins during mouse liver development. For the sake of simplicity, distribution of Types I and III collagen, fibronectin, and WGA-binding sites are not depicted. HSPG, heparan sulfate proteoglycan; PNA, peanut agglutinin.

Liver Primordium Formation.

In embryonic development, the hepatocardiac mesoderm induces the ventral endoderm of the foregut to differentiate into the hepatic endoderm and then hepatoblasts.19, 20 During these inductive periods, the hepatic endoderm constructs a diverticulum, and the hepatic cords from the diverticulum, which consist of hepatoblasts, invade the subjacent mesenchyme. Acidic or basic fibroblast growth factors or bone morphogenetic proteins may be involved in such induction.20, 27, 28 Hepatic cords in the parts distal to the hepatic diverticulum were not decorated with basal laminar components of laminin, type IV collagen, nidogen, or HSPG. In contrast, the cells close to the hepatic diverticulum, which faced the foregut lumen, possessed the basal lamina like other foregut endodermal cells. This implies that the basal lamina is not prerequisite to the invasion of hepatic cords into the subjacent mesenchyme, which has been shown in other organogenesis and cancer invasion.29–31 There is also evidence that the deposition of the basal lamina may not be favorable to hepatoblast or hepatocyte differentiation, based on an inactivation study of the Prox1 gene.23 It should also be noted that the hepatic endoderm lacked the expression of the α6 subunit of integrin and poorly expressed the β1 subunit of integrin, both of which were strongly or moderately detected in other foregut endodermal cells. These expression patterns were exclusive to that of α-fetoprotein messenger RNA (data not shown). Because the septum transversum mesenchyme induces the formation of the diverticulum in the hepatic endoderm and subsequent invasion of the hepatic cords, factors from the mesenchyme may act on the endoderm to reduce the expression of both α6 and β1 integrin subunits. Their reduced expression may be coupled with the morphogenesis of the liver primordium such as invasion of the hepatic cords.

Sinusoid Development.

The present study demonstrated that the omphalomesenteric veins could branch in the septum transversum mesenchyme without the invasion of hepatic cords, which could lead to the formation of sinusoids or portal veins. Their interactions with hepatic cords might not be required for such elaborate branching. We also found that blood vessel formation occurred subjacent to the hepatic endoderm and the hepatic cords from the α6 integrin subunit immunostaining in 9.5-day liver primordium. This result agrees well with the PECAM-1 staining reported by Matsumoto et al.22 The factors from these blood vessels can work on the hepatic endodermal cells to augment their proliferation. The endothelial cells of the sinusoids could also be derived from the septum transversum mesenchyme, supporting the idea of Severn.14

Our immunostaining of basal laminar components, the α6 integrin subunit, and PECAM-1 revealed that prospective vascular structures (probably primitive sinusoidal structures), which often possessed small lumina, existed between hepatic cords associated with hematopoietic cells from early development of fetal livers (see Fig. 8). α6 Integrin subunit immunoreactivity in endothelial cells of the sinusoidal structures was different from endothelial cells of portal and hepatic veins, which were comparatively weakly immunostained at young stages for this protein. Double immunofluorescent analyses of the α6 integrin subunit, desmin, laminin, and cytokeratins demonstrated that the primitive sinusoidal structures were surrounded by desmin-positive stellate cells, and that the basic cell arrangement of hepatic cords, stellate cells, and sinusoidal structures, which is seen in the adult liver, developed with the commencement of liver development. Free hematopoietic cells, which were associated with those sinusoidal structures and were α6 integrin subunit-positive, also possibly gave rise to sinusoidal endothelial cells. Granular immunostaining of basal laminar components in these structures may suggest that they are very immature. Because each basal laminar component colocalized in these structures, granular insoluble matrices may play an important role in their development. Sinusoidal antigens develop in midgestation stages in rat fetuses.32

Functional sinusoid development was accompanied by changes in immunolocalization of basal laminar components. During fetal development, granular immunostaining of basal laminar components became more continuous. Nidogen and laminin immunostaining decreased in the sinusoidal structures at perinatal stages. In 2-week-old mouse livers, adult-type sinusoidal structures were established as seen by basal laminar and WGA staining. These changes may be involved in both sinusoid and hepatocyte maturation.

Formation of Biliary System.

Epithelial cells of extrahepatic bile ducts such as the common bile duct and cystic duct possessed the basal lamina, which was decorated by laminin, nidogen, HSPG, and type IV collagen from the beginning of their formation (see Fig. 8). These cells also strongly expressed some integrin subunits, whereas progenitors of intrahepatic bile duct cells did not express those molecules. These results clearly highlight the different expression pattern of ECM components and integrins in the extrahepatic bile duct cells from that in the intrahepatic bile duct cells, suggesting that they have different origins. The development of the hepatic ducts, which connect the extrahepatic bile ducts with the intrahepatic bile ducts, were intermediate between both ducts in expression of basal laminar components and integrin subunits (see Fig. 8). Because epithelial cells of the extrahepatic bile ducts also transiently expressed α-fetoprotein messenger RNA as in hepatic and intrahepatic bile duct cells,1 they could also be derived from hepatoblasts. In hepatoblast populations, the differentiation of the biliary duct system by hepatoblasts may proceed progressively (i.e., the formation of the extrahepatic bile duct, hepatic ducts, and then intrahepatic bile ducts). Because these extrahepatic and intrahepatic bile ducts were not confluent along portal veins in the beginning, elaborate mechanisms of connecting these discontinuous progenitors must work later.

We have shown that intrahepatic biliary differentiation proceeds in a stepwise way by cytokeratin isoform expression in fetal rat livers.1, 2 In the present study, further detailed sequences of bile duct differentiation were demonstrated. All basal laminar components, which mostly colocalized in the periportal area, were seen at almost the same time: 13.5 or 14.5 days' gestation (see Fig. 8). PNA-binding sites appeared in biliary cells after 14.5 days' gestation. Expression of α3, α5, and α6 subunits and the β4 subunit of integrin commenced in some biliary progenitors after 14.5–15.5 days and 15.5 days, respectively. These stages coincide with that of cytokeratin 19 expression.1, 5, 6 Cytokeratin 7 and Dolichos biflorus agglutinin-binding sites appear to be more progressive markers.1, 4 Differentiated biliary epithelial cells strongly expressed cellular receptors for basal laminar components, which may suggest their establishment as a typical epithelial tissue. Sequences of basal laminar deposition and integrin subunit expression in biliary progenitors also indicated that after basal laminar deposition occurred, their cellular receptors were highly induced in intrahepatic bile duct differentiation. Nidogen may be a key molecule for the bile duct differentiation, because this molecule stabilizes the binding of laminin and type IV collagen.33

Although bile duct differentiation occurs only in periportal areas, histological differences are not clear between portal veins and hepatic veins at early stages (11.5 and 12.5 days); however, after 13.5 days, the development of periportal connective tissue distinguishes the veins.15, 34 Our immunostaining demonstrated stronger immunoreactivities of α2, α3, and β4 integrin subunits in endothelial cells of portal veins, and of α3 integrin subunits in periportal connective tissue, which may be related to biliary cell differentiation. It is also noteworthy that at 13.5 and 14.5 days, expression of laminin and nidogen appeared comparatively even in the whole liver parenchyma, and by 17.5 days, differences of laminin and nidogen staining became more obvious between portal and hepatic veins, being consistent with biliary cell differentiation. In midgestation stages, the whole liver parenchyma may be equivalent for bile duct differentiation, during which the signals for portal bile duct differentiation may act on periportal hepatoblasts.

Hepatocyte Maturation and ECM.

The immunolocalization of ECM components around hepatoblasts and hepatocytes changed from granular to continuous staining as indicated in sinusoid development. Laminin and nidogen staining decreased in sinusoidal areas, especially in postnatal development. Hepatocyte morphology and maturation proceeded with these changes of ECM components. Several ECM components can control hepatocyte maturation and morphology in their primary culture.35, 36 In granular immunostaining of basal laminar components, several components such as laminin and nidogen colocalized, which suggests that they function as anchorage for hepatoblasts and hepatocytes. Hepatoblasts and hepatocytes were not reactive with the anti-integrin antisera examined, suggesting that they may express very small amounts of these integrin subunits or other subtypes. Hepatoblasts or hepatocytes have different repertoires from biliary epithelial cells with regard to integrin expression.


We thank Professor Emeritus Takeo Mizuno of the University of Tokyo and Professor Nelson Fausto of the University of Washington for their interest in our study and their encouragement. We also thank Mr. Kim Barrymore for his help in preparing the manuscript.