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Molecular and Cellular Biology, August 2006, p. 6149-6156, Vol. 26, No. 16
0270-7306/06/$08.00+0     doi:10.1128/MCB.00298-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Crucial Role of the Small GTPase ARF6 in Hepatic Cord Formation during Liver Development

Teruhiko Suzuki,1,2 Yoshiakira Kanai,3 Takahiko Hara,4 Junko Sasaki,8 Takehiko Sasaki,8 Michinori Kohara,5 Tomohiko Maehama,6 Choji Taya,7 Hiroshi Shitara,7 Hiromichi Yonekawa,7 Michael A. Frohman,9 Takeaki Yokozeki,1 and Yasunori Kanaho1*

Department of Physiological Chemistry, Graduate School of Comprehensive Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Ten-nohdai, Tsukuba 305-8575, Japan,1 Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongoh, Bunkyo-ku, Tokyo 113-0033, Japan,2 Department of Veterinary Anatomy, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1 Yayoi Bunkyo-ku, Tokyo 113-8657, Japan,3 Stem Cell Project,4 Infectious Diseases Project,5 Biomembrane Signalling Project,6 Core Technology & Research Center, Laboratory of Mouse Model for Human Heritable Diseases, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan,7 Department of Pathology and Immunology, Akita University School of Medicine, Akita 010-8543, Japan,8 Center for Developmental Genetics and Department of Pharmacology, Stony Brook University, New York, New York 11794-51409

Received 17 February 2006/ Returned for modification 28 March 2006/ Accepted 3 June 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mammalian small GTPase ADP-ribosylation factor 6 (ARF6) plays important roles in a wide variety of cellular events, including endocytosis, actin cytoskeletal reorganization, and phosphoinositide metabolism. However, physiological functions for ARF6 have not previously been examined. Here, we described the consequence of ARF6 ablation in mice, which manifests most obviously in the context of liver development. Livers from ARF6–/– embryos are smaller and exhibit hypocellularity, due to the onset of midgestational liver cell apoptosis. Preceding the apoptosis, however, defective hepatic cord formation is observed; the liver cells migrate abnormally upon exiting the primordial hepatic epithelial sheet and clump rather than becoming dispersed. Consistent with this observation, the ability of hepatocyte growth factor/scatter factor (HGF) to induce hepatic cord-like structures from ARF6–/– fetal hepatocytes cultured in vitro in collagen gel matrix is impaired. Finally, we show that endogenous ARF6 in wild-type fetal hepatocytes is activated in response to HGF stimulation. These results provide evidence that ARF6 is an essential component in the signaling pathway coupling HGF signaling to hepatic cord formation.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The mammalian small GTPase ADP-ribosylation factor (ARF) family consists of six related gene products, ARF1 to ARF6, which have been divided into three classes based on sequence homology (27). Class I contains ARF1 to ARF3, class II ARF4 and ARF5, and class III ARF6. Classes I and II of ARFs localize primarily to intracellular organelles, including the Golgi apparatus, and have been implicated in many types of intracellular membrane vesicle trafficking events, e.g., vesicular transport between the endoplasmic reticulum and the Golgi and receptor recycling from endosomes to the plasma membrane (5, 9, 26, 28). In contrast, ARF6, the sole member of class III, localizes to the plasma membrane and has been well documented as playing a crucial role in endocytosis (2, 3, 6, 7, 17). In addition, we and others have reported that ARF6 uniquely regulates membrane morphologies of nonneuronal and neuronal cells through the activation of the lipid messenger-producing enzyme, phosphatidylinositol 4-phosphate 5-kinase, which reorganizes the actin cytoskeleton through its product phosphatidylinositol 4,5-bisphosphate (8, 9). From these reports, it could be speculated that ARF6 regulates physiological functions that depend on morphogenetic changes regulated via actin cytoskeletal reorganization. Although the functions for ARF6 described above have been extensively characterized at the cellular and molecular levels, the requirement for ARF6 physiologically has not yet been determined.

To address this issue, we have generated and analyzed ARF6 knockout mice. The results obtained in this study revealed that ARF6–/– embryos exhibit abnormal liver development characterized by reduced size and aberrant structure, due to defective hepatic cord formation, and we identify a specific signaling pathway that appears to be responsible for the abnormal development.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Targeted disruption of the ARF6 gene. A mouse 129/Ola genomic library generously provided by J. Penninger was screened and a clone containing the ARF6 gene isolated. An 8.3-kb DNA fragment generated by the digestion of the clone with ApaI and a 691-bp fragment generated by PCR using 5'-ATACTTTAGCGGCCGCCTGTCTGACACACTCATGT-3'and 5'-TTGTCTAGACCGGAAGGAGAGAAATCCAA-3'as primers were used as the long and short arms of the targeting vector, respectively (Fig. 1A). The targeting vector, which replaced the fragment encompassing the first ATG of ARF6 with a neomycin-resistance cassette (Neo) and contained arms flanking Neo, was constructed, linearized, and electroporated into E14K embryonic stem (ES) cells, also generously provided by J. Penninger. ES cell colonies resistant to G418 were screened for homologous recombination by PCR using primers specific for the ARF6 genomic sequence and Neo (ARF6 primer, 5'-CTTGTTCTAGGCGGCAGTTA-3'; Neo primer, 5'-CCTACCGGTGGATGTGGAAT-3'). Two independent heterozygous ES cell clones were used to generate chimeric mice by injection into blastocysts from C57BL/6 mice. Chimeric male mice were crossed with C57BL/6 females, and the ARF6+/– mice obtained were mated to obtain ARF6-null mice. The phenotypes reported in this study were observed in both lines. Genotyping was carried out by Southern blotting or PCR analyses. For Southern blotting, genomic DNA was digested with EcoRV and detected with a 3' probe flanking the targeting construct as described for Fig. 1A. The probe was generated by PCR amplification using 5'-GCAGTGTCAGCCATTAACGT-3' and 5'-CTACTGGTCTTAAGACATTTG-3' as primers. For PCR genotyping, 5'-CCTACCGGTGGATGTGGAAT-3', 5'-TTCAAAAAGAGAGTGGCAATTCA-3', and 5'-AGGAGCTGCACCGCATTATC-3'were used as primers.


Figure 1
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  		FIG. 1. Targeted disruption of the ARF6 gene. (A) Schematic representation of ARF6 targeting. The genomic structure of the ARF6 gene is shown at the top. Noncoding regions of exons are represented by filled boxes, and the open reading frame is represented by the hatched box. RV, EcoRV; A, ApaI. (B to D) Genotypes were verified by Southern blotting (B), PCR (C), and Western blotting of total lysates prepared from embryonic fibroblasts of each genotype (D).

 
All mice employed in this study were maintained in a pathogen-free facility of the Tokyo Metropolitan Institute of Medical Science, and the experimental protocols for the animal studies were approved by the Ethics Review Committee for Animal Experimentation of the Institute.

Antibodies, chemicals, and probes. Anti-albumin antibody was purchased from Bethyl Laboratories, anti-Liv2 from MBL, anti-c-Met from Santa Cruz Biotechnology, Texas Red-conjugated streptavidin from Perkin Elmer Life Sciences, hepatocyte growth factor (HGF) from Sigma-Aldrich, anti-Ter119 and anti-CD45 from BD Pharmingen, anti-active caspase 3 from Cell Signaling, and rhodamine-phalloidin from Molecular Probes. A polyclonal anti-ARF6 antiserum was a generous gift of J. G. Donaldson.

In situ hybridization and histochemistry. To synthesize a probe to detect ARF6 mRNA expression, an ARF6 cDNA, a generous gift of K. Nakayama, was digested with HincII, subcloned into pBluescript II SK(+), and then digested with BglII. Using the digested plasmid, a digoxigenin-labeled antisense probe corresponding to nucleotides 507 to 992 of the ARF6 gene was generated using a DIG RNA-labeling kit (Roche). Whole-mount in situ hybridization was carried out as described previously (12), and the stained embryos were sectioned horizontally to analyze ARF6 mRNA expression in the liver.

For immunohistochemical analysis, embryos were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), embedded in optimal cutting temperature compound (Sakura Finetechnical), sectioned, and stained with antibodies. Bromodeoxyuridine (BrdU)-positive cells were detected using a BrdU in situ detection kit (BD Pharmingen) according to the manufacturer's protocol. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was performed using the DeadEnd fluorometric TUNEL system (Promega) according to the manufacturer's protocol. For double staining of hepatocytes and erythroid cells with their specific antibodies (the anti-albumin and Ter119 antibodies, respectively) and TUNEL, the anti-albumin and anti-Ter119 antibodies were detected using biotin-conjugated secondary antibodies, and visualization was performed using Texas Red-conjugated streptavidin. To enhance the anti-albumin and anti-Liv2 antibody signals, a VECTASTAIN Elite ABC kit (Vector Laboratories) was used.

Culture of fetal hepatocytes. Livers dissected from embryonic day 13.5 (E13.5) wild-type and ARF6–/– embryos were minced and liver cells dissociated by being sequentially incubated in liver perfusion medium and liver digest medium (Invitrogen). Dissociated cells were seeded on collagen-coated dishes or cover glasses and incubated for 2 h in the basic medium, which consists of Eagle minimum essential medium, 1 mM sodium pyruvate, nonessential amino acid solution, 500 U/ml penicillin, 50 µg/ml streptomycin, and 0.1 µM dexamethasone, supplemented with insulin-transferrin-selenium -X and 10% fetal calf serum (FCS), during which time the fetal hepatocytes attached. The cells were then washed with the basic medium to remove hematopoietic cells and dead cells and further incubated overnight in the basic medium supplemented with 10% FCS. The cells were again washed with the basic medium to completely remove the remaining hematopoietic cells. The purity of fetal hepatocytes thus obtained was more than 90%, as assessed by immunostaining using the anti-albumin antibody.

Assay of hepatic cord-like structure formation of fetal hepatocytes. The HGF-induced cord-like structure formation assay was performed as previously reported (13) with minor modifications. Briefly, fetal hepatocytes cultured on collagen-coated dishes were detached with liver perfusion medium and liver digest medium and replated on 1.2 mg/ml collagen gel. After 2 h, the attached cells were washed, overlaid with collagen gel, and incubated in the basic medium supplemented with 10% FCS for 24 h. The cells were then incubated without or with 25 ng/ml of HGF in the basic medium supplemented with 10% FCS for 24 h. After fixation with 4% paraformaldehyde in PBS, the cells were observed using light microscopy. To assess the morphologies of the colonies and to count the number of cells in each colony, fixed cells were stained for actin and nuclei using rhodamine-phalloidin and DAPI (4',6'-diamidino-2-phenylindole), respectively. Colonies composed of fewer than 10 cells were assessed. Colonies that formed cord-like structures in response to HGF stimulation were defined as those elongated more than 70 µm on the long axis, with protrusions at the tips of the colonies. Under nonstimulated conditions, fewer than 25% of the colonies exhibited extension defined as such.

Pulldown assay of activated ARF6. To analyze ARF6 activation, ARF6-GTP pulldown assays were carried out as previously reported (20). Briefly, 5 x 105 fetal hepatocytes seeded on collagen-coated dishes in the basic medium were cultured overnight. After being washed, fetal hepatocytes were further incubated for 4 h and then stimulated with or without 25 ng/ml HGF for 2 min. Cells were harvested and lysed using a lysis buffer composed of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM MgCl2, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1% Triton X-100, 10% glycerol, 1 µg/ml aprotinin, and 1 µg/ml leupeptin. Lysate supernatants were mixed with glutathione-Sepharose beads preconjugated with glutathione S-transferase-GGA31-226 and incubated for 30 min with gentle rotation. The beads were washed three times with the washing buffer composed of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM MgCl2, 1% NP-40, 10% glycerol, 1 µg/ml aprotinin, and 1 µg/ml leupeptin, and the ARF6-GTP bound to the glutathione S-transferase-GGA31-226 beads eluted using sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer. The eluted, active form of ARF6 was detected by Western blotting with anti-ARF6 antiserum.

Hematopoietic cell culture. Hematopoietic cells prepared from E13.5 embryonic livers were cultured in StemPro34 supplemented with mouse stem cell factor (100 ng/ml), interleukin-3 (10 ng/ml), and interleukin-6 (10 ng/ml). After 3 days, cells were harvested and stained with annexin V-fluorescein isothiocyanate (FITC) (BD Pharmingen) according to the manufacturer's protocol. To evaluate the extent of apoptosis, the ratios of annexin V-positive cells to propidium iodide (PI)-negative cells were analyzed by flow cytometry.

Proliferation assay of fetal hepatocytes. Proliferation of cultured fetal hepatocytes was analyzed by [3H]thymidine incorporation. Fetal hepatocytes were seeded on collagen-coated dishes and cultured overnight in the presence or absence of 25 ng/ml HGF in the medium consisting of minimum essential medium, 1 mM sodium pyruvate, nonessential amino acid solution, 500 U/ml penicillin, and 50 µg/ml streptomycin. Cells were extensively washed and further cultured for 6 h in the presence of 5 µCi/ml [3H]thymidine. Cells were then fixed with 10% trichloroacetic acid for 30 min on ice. After being washed with PBS, samples were treated with 1 N NaOH for 20 min at 37°C followed by 1 N HCl treatment for neutralization. Samples thus prepared were harvested, and the [3H]thymidine incorporated was measured by liquid scintillation counting.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Targeted disruption of ARF6 gene causes liver developmental defect. To investigate the physiological function of ARF6, we targeted the ARF6 gene by homologous recombination using a targeting vector that replaced a large part of the protein sequence, including the first methionine, with a neomycin resistance cassette (Fig. 1A). Disruption of the ARF6 gene was verified by Southern blotting, PCR, and Western blotting (Fig. 1B to D). Heterozygous targeted mice (ARF6+/–) were healthy and fertile. However, homozygous targeted mice (ARF6–/–) exhibited almost completely penetrant embryonic lethality, starting at midgestation and continuing through birth (Table 1). Visual and anatomical inspection of the ARF6–/– embryos revealed several abnormalities, including edema, which was frequently observed at the head (Fig. 2A to C), and occasional hemorrhage and anemia in the mid- to late-gestational periods (data not shown). In addition, a dramatic decrease in the size of the liver was readily apparent (Fig. 2A to H), but overt abnormalities in other organs were not (data not shown). Total body weights for the ARF6–/– embryos did not significantly differ from those of ARF6+/+ or ARF6+/– littermates (Fig. 2I); however, the ratios of liver weights to body weights were substantially lower in the ARF6–/– embryos (5.3% ± 0.7%, 5.3% ± 0.6%, and 2.9% ± 0.7% [means ± standard deviations {SDs}] for ARF6+/+, ARF6+/–, and ARF6–/– embryos, respectively) (Fig. 2J). In addition to their small sizes, the livers dissected from the ARF6–/– embryos exhibited aberrant structures characterized by shriveled and hypoplastic lobes, although they had the correct number of lobes. The lobes appeared to be thinner and paler than those of wild-type littermates (Fig. 2D to H); the paleness might be attributable to their thinness. These results suggest that ARF6 is essential for liver development, consistent with the observation that ARF6 is highly expressed in the livers of E10.5 embryos (Fig. 2K to O).


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  		TABLE 1. Genotypes of progeny

 

Figure 2
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  		FIG. 2. Defective liver formation in ARF6 knockout embryos. (A to C) Appearance of E13.5 embryos. Note the reduction in the size of the ARF6–/– embryonic liver (C, black arrowhead). The white arrowhead in panel C indicates edema at the head. Scale bars, 1 mm. (D to H) Livers dissected from E13.5 embryos. Images shown are views of embryonic livers from the top (D and E), bottom (F), and side (G and H). Note the aberrant structures of the lobes in the ARF6–/– livers as well as the reductions in size (E, F, and H). White arrowheads in panels F, G, and H indicate the outlines of liver lobes. Scale bars, 1 mm. (I) Body weights of E13.5 embryos. Data shown are the means ± SDs. +/+, n = 12; +/–, n = 21; –/–, n = 9. (J) The ratios of liver weight to body weight for E13.5 embryos. Data shown are the means ± SDs. +/+, n = 12; +/–, n = 21; –/–, n = 9. An asterisk denotes statistical significance (P < 0.005). (K) Whole-mount in situ hybridization analysis of E10.5 ARF6+/+ (+/+, left image) and ARF6–/– (–/–, right image) embryos using an antisense ARF6 probe. The arrowhead indicates the liver, which stained intensely with the probe. Scale bar, 1 mm. (L and M) Higher-magnification images of the whole-mount-in situ-hybridized embryos shown in panel K. Scale bars, 100 µm. (N and O) Sections of whole-mount-in situ-hybridized E10.5 ARF6+/+ (N) and ARF6–/– (O) embryos using an antisense ARF6 probe. Scale bars, 100 µm. L, liver; Fl, forelimb bud; G, gut.

 
ARF6–/– embryonic livers exhibit progressive apoptosis. Hematoxylin and eosin staining analysis revealed that ARF6–/–E13.5 embryonic livers have a sponge-like, hypocellular structure, especially in peripheral regions (Fig. 3A and B), which could also at least in part explain the paler livers of ARF6–/– embryos as described above. Three primary hypotheses could account for the observed hypocellularity and smaller sizes of the livers: (i) defective proliferation of liver cells, (ii) defective colonization of hematopoietic cells into the livers, and (iii) progressive apoptosis of liver cells. The first possibility appears unlikely, since the ratios of proliferating cells to total cells in the livers as assessed by BrdU incorporation were not significantly decreased, even in the peripheral regions (Fig. 3C to F) (although BrdU-positive cells are reduced in number at the peripheries of the ARF6–/– embryonic livers, the total cell numbers are reduced in parallel). Defective colonization of hematopoietic cells was also not evident: flow cytometric analysis revealed that the ratios of hematopoietic cells (Ter119+ or CD45+ cells) to total liver cells in ARF6–/– embryonic livers (84.0% ± 3.2% [mean ± SD], n = 4) were not statistically different from those observed for wild-type embryonic livers (85.6% ± 0.9% [mean ± SD], n = 4). However, E13.5 ARF6–/– embryonic livers were highly noteworthy for a marked increase in apoptosis (Fig. 3G to L) and significant activation of caspase 3 (Fig. 3M) as detected by TUNEL staining and by Western blotting with the anti-active caspase 3 antibody, respectively. In contrast, no activation of caspase 3 was observed in ARF6–/– embryonic heart (Fig. 3M), indicating that the induction of apoptosis was tissue specific. During mid- to late gestation, the embryonic liver is composed primarily of hepatocytes and hematopoietic cells (11). To determine the lineage specificity of the apoptotic cells, TUNEL staining was combined with immunodetection of albumin and Ter119 as markers, respectively, of hepatocytes and erythroid cells, the major lineage of hematopoietic cells in the fetal liver. Apoptosis was observed for both cell types (Fig. 4A and B), indicating that the reduction in size and the hypocellularity of ARF6–/– embryonic liver are attributable to progressive apoptosis of multiple cell lineages. Interestingly, however, enhanced apoptosis was not observed at an earlier stage (E10.0) of development (Fig. 4C and D), nor did primary cultured ARF6–/– fetal hepatocytes or hematopoietic cells prepared from E13.5 embryos show enhanced apoptosis (Fig. 4E and F). Taken together, these findings suggest that the observed progression of apoptosis is a context-specific response in the embryonic liver niche rather than a cell type-specific or cell-intrinsic phenomenon.


Figure 3
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  		FIG. 3. Hypocellularity and induction of apoptotic cell death in the ARF6–/– embryonic liver. (A and B) Hematoxylin and eosin staining of liver sections prepared from E13.5 wild-type (Wt) and ARF6–/– embryos. Note the hypocellularity in the ARF6–/– embryonic liver. (C to F) BrdU-incorporated cells in E13.5 wild-type (C and E) and ARF6–/– (D and F) embryonic livers were detected with anti-BrdU antibody (brown) and counterstained with hematoxylin (blue). The boxed areas in panels C and D are shown at higher magnification in panels E and F, respectively. (G to L) Liver sections prepared from E13.5 wild-type and ARF6–/– embryos were stained with PI (to visualize nuclei [G and H]) and TUNEL (as an indicator of apoptosis [I and J]). The boxed areas in panels G and H were merged to present both channels (K and L). PI staining is visualized as red and TUNEL-positive cells as green. (M) Activation of caspase 3 in E13.5 embryonic livers and hearts was analyzed by Western blotting of total lysates with the anti-active caspase 3 antibody. Scale bars, 100 µm (A to D and G to J) and 50 µm (E, F, K and L).

 

Figure 4
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  		FIG. 4. Analysis of apoptotic cell death in ARF6–/– embryonic liver. (A and B) E13.5 ARF6–/– embryonic liver sections were double stained with TUNEL (green) and antialbumin (to identify hepatocytes [red]) (A) or anti-Ter119 antibodies (to identify erythroid cells [red]) (B). TUNEL-positive and -negative cells are indicated by arrowheads and arrows, respectively. (C and D) Liver sections prepared from E10.0 wild-type (Wt) (C) and ARF6–/– (D) embryos were double stained with TUNEL (green) and PI (red). Arrowheads represent TUNEL-positive liver cells, and dotted lines show the outlines of the livers. (E) In vitro-cultured fetal hepatocytes prepared from E13.5 embryonic livers were stained with TUNEL. TUNEL-positive cells were counted to calculate the percentages of apoptotic cells (n = 3). Data shown are the means ± SDs. (F) Hematopoietic cells prepared from E13.5 embryonic livers were cultured for 3 days. To evaluate the extent of apoptosis, the harvested cells were stained with annexin V-FITC and the percentages of annexin V-positive cells in PI-negative cells assessed by flow cytometry (n = 3). Data shown are the means ± SDs. G, gut; L, liver; scale bars, 10 µm (A and B) and 50 µm (C and D).

 
Defective hepatic cord formation in ARF6–/– embryonic liver. To identify the underlying primary defect in ARF6–/– embryonic liver preceding the terminal apoptotic event, ARF6–/– embryonic livers at various stages of development were immunohistochemically analyzed using anti-Liv2 and anti-albumin antibodies to identify precursor cells at earlier stages of hepatogenesis (29) and hepatocytes, respectively. At E9.0, the liver diverticula are characterized by a series of continuous epithelial sheets. Similar patterns of Liv2 staining were observed in wild-type and ARF6–/– liver diverticula (Fig. 5A and B), suggesting that primary hepatoblast expansion and differentiation are not impaired by ARF6 deficiency. By E10.0, the epithelial sheets have undergone extensive elongation and branching into hepatic cord structures (Fig. 5C). Strikingly, however, such structures were not observed in ARF6–/– embryonic livers; instead, the Liv2-positive cells remained clustered at the terminal edges of the epithelial sheets (Fig. 5D). Importantly, this E10.0 anatomical abnormality precedes the initiation of apoptosis (Fig. 3J and 4D) and the other defects described above. Aberrant architecture characterized by hepatocyte clustering was similarly observed at E11.5 and E13.5 in ARF6–/– embryos (Fig. 5F and H), although the severity of hepatic cord formation defect varied among the ARF6–/– embryos (data not shown). In contrast, hepatocytes of wild-type embryos dispersed into well-elongated and -branched cord structures (Fig. 5E and G). These findings suggest that the primary defect caused by ARF6 deficiency involves a failure of hepatic cord formation rather than a direct pathway to apoptosis.


Figure 5
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  		FIG. 5. Defective hepatic cord formation in ARF6–/– embryonic liver. (A to H) Liver sections prepared from wild-type (Wt) (left panels) and ARF6–/– (right panels) embryos at E9.0 (A and B), E10.0 (C and D), E11.5 (E and F), and E13.5 (G and H) were stained with anti-Liv2 (A to D) and antialbumin (E to H) antibodies. LD, liver diverticulum; G, gut; scale bars, 50 µm.

 
ARF6–/– fetal hepatocytes cultured in vitro in collagen gel matrix exhibit defective hepatic cord-like structure formation in response to HGF stimulation. Targeted inactivation of HGF, also known as scatter factor (21), phenocopies the reduction in the liver size and the gross aberrant fetal liver development observed here for ARF6–/– mice. Moreover, ARF6 has been reported to be involved in the HGF-dependent signaling pathway in MDCK cells (14, 15). These observations led us to investigate this possibility, and we found accordingly that ARF6 is activated by HGF stimulation in cultured fetal hepatocytes prepared from wild-type embryos (Fig. 6A). To examine the relevance of this pathway for the ARF6–/– phenotype, an in-culture model for hepatic cord formation (13) was then used. As previously reported (13), wild-type fetal hepatocyte colonies cultured in collagen gel formed hepatic cord-like structures in response to HGF stimulation (Fig. 6B). In contrast, cord-like structure formation was significantly impaired in fetal hepatocyte colonies prepared from ARF6–/– embryonic liver (Fig. 6B). This was not due to a reduction of the HGF receptor, c-Met, since the expression level of c-Met in ARF6–/– fetal hepatocytes was comparable to that observed for wild-type fetal hepatocytes, as detected by Western blotting using an anti-c-Met antibody (data not shown). To quantify the cord-like structure formation, fetal hepatocytes were cultured at lower density to generate individual colonies. Under these conditions, 61.5% ± 1.0% (mean ± standard error of the mean [SEM]) of fetal hepatocyte colonies prepared from wild-type liver formed cord-like structures in response to HGF stimulation, whereas fewer colonies with cord-like structure were observed for hepatocytes prepared from ARF6–/– embryos (45.6% ± 1.8% [mean ± SEM]) (Fig. 6C). In contrast, HGF-dependent proliferation of ARF6–/– fetal hepatocytes was comparable to that of wild-type fetal hepatocytes (Fig. 6D). These findings, taken together, demonstrate that ARF6 plays a crucial role specifically in the HGF-dependent signaling pathway coupled to hepatic cord formation.


Figure 6
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  		FIG. 6. Involvement of ARF6 in HGF-induced hepatic cord-like structure formation. (A) Activation of ARF6 by HGF stimulation of cultured fetal hepatocytes (n = 3). (B) Suppression of HGF-induced cord-like structure formation in ARF6–/– fetal hepatocytes. Scale bar, 100 µm. (C) Quantification of the HGF-induced cord-like structure formation of fetal hepatocyte colonies. Data shown are the means ± SEMs (n = 4). The asterisk denotes statistical significance (P < 0.005). (D) Effect of ARF6 deficiency on HGF-induced proliferation of fetal hepatocytes, as assessed by [3H]thymidine incorporation. Data shown are the means ± SDs (n = 3).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study, showing that disruption of the ARF6 gene leads to almost complete lethality starting at midgestation (Table 1), demonstrates that ARF6 is essential for mouse development. The most obvious defect of ARF6–/– embryos is in liver development. The primary effect of ARF6 deficiency on liver development appears to be interference with hepatic cord formation (Fig. 5). This observation is supported by the successful modeling of the abnormal phenotype in an in vitro culture system for hepatic cord-like structure formation as induced by HGF using fetal hepatocytes (Fig. 6B). Moreover, ARF6 in cultured fetal hepatocytes is activated in response to HGF stimulation (Fig. 6A). Collectively, these results lead us to conclude that ARF6 functions at least in part as a signaling molecule in the HGF-dependent signaling pathway coupled to hepatic cord formation.

Although the molecular mechanism by which ARF6 regulates the cascade of signaling required for hepatic cord formation remains to be clarified, it is plausible that ARF6 functions by regulating membrane morphology and/or cell migration through controlling actin cytoskeletal reorganization, which is a well-known role for it in model systems (1, 6, 9, 16, 18). This idea is derived from the observation that elongation and branching of the E10.0 hepatic epithelial sheets were abnormal in the ARF6–/– embryonic liver (Fig. 5D). Alternatively, membrane trafficking regulated by ARF6 might also be critical for hepatic cord formation, as suggested by reports that ARF6-regulated internalization of E-cadherin enhances the motility of epithelialized cells (14) and that the level of E-cadherin decreases in migrating fetal hepatocytes during liver development (24).

Disruption of HGF-dependent cord-like structure formation in ARF6–/– fetal hepatocytes in the in vitro assay system was not complete (Fig. 6B and C), suggesting that HGF utilizes another signaling system, as well as the ARF6-mediated signaling pathway, to couple HGF signaling to cord-like structure formation. If this is true and HGF is the critical physiological factor for hepatic cord formation, then disruption of hepatic cord formation in ARF6–/– embryos should be less severe than that in HGF knockout embryos. However, the severity of the liver developmental defect in ARF6–/– embryos was almost the same as that reported for HGF knockout embryos (21). These observations lead us to speculate that, in addition to HGF, other hepatic cord formation-promoting factors most likely exist. Supporting this assumption, it has previously been reported that epidermal growth factor and transforming growth factor ß also induce hepatic cord-like structure formation in vitro (13, 19). In addition, it has been reported that transforming growth factor ß type III receptor-deficient mouse embryos display defects in the ultrastructure of the liver (25). Finally, previous reports demonstrating that ARF6 is absolutely required for these types of growth factor-induced cell functions in other settings (4, 9) also support the hypothesis described above.

In the present study, we demonstrated that the smaller sizes and hypocellularity of the ARF6–/– embryonic livers were attributable to the progression of liver cell apoptosis (Fig. 3G to M); proliferation of fetal hepatocytes in vitro and in vivo was not impaired by ARF6 deficiency (Fig. 3C to F and 6D), inconsistent with prior reports that ARF6 is essential for cytokinesis (22, 23). This apparent discrepancy suggests the existence of compensatory or redundant mechanisms that promote cytokinesis not only in hepatocytes but in most if not all other cell types. Moreover, we would emphasize that the induction of apoptosis in the ARF6–/– embryonic liver, which is not cell lineage specific (Fig. 4A and B), does not seem to be the primary consequence of ARF6 deficiency. This conclusion is supported by the observation that abnormal liver architecture (Fig. 5C and D), but not apoptosis (Fig. 4C and D), was observed at earlier stages (E10.0) of embryonic liver development, and cultured ARF6–/– fetal hepatocytes or hematopoietic cells prepared from E13.5 embryos did not exhibit increased apoptosis (Fig. 4E and F). Instead, we would propose that the induction of apoptosis of fetal hepatocytes and of hematopoietic cells that would normally come to coreside in the cord niche might be attributable secondarily to an aberrant fetal liver microenvironment that is unsupportive for liver cell survival, due to incomplete hepatic cord formation. Such an aberrant microenvironment could cause the activation of caspase 3, resulting in the induction of apoptosis, although the details of this death response remain to be elucidated.

ARF6–/– embryos were frequently found to be anemic, which might be responsible for the mid- to late-gestational lethality (data not shown). Considering that we observed progressive and substantial apoptosis of hematopoietic cells in the embryonic liver (Fig. 4), it is quite reasonable to speculate that anemia caused in ARF6–/– embryos is attributable to progressive apoptosis of hematopoietic cells that is triggered by the defect of liver formation, as was suggested for HGF knockout mice (21). We cannot rule out, however, an additional effect on hematopoiesis as well. Nonetheless, the lethality observed for ARF6–/– embryos during mid- to late gestation appears attributable to the observed defects in liver formation. This idea is consistent with the observation that there was some variation in the stage of lethality of ARF6–/– embryos (Table 1), since variation was also observed in the severity of the liver formation defect (data not shown).

In conclusion, this report provides evidence for the first time that ARF6 physiologically functions in liver development by regulating hepatic cord formation. In addition to this function, ARF6 may also be involved in other physiological and pathological events, such as development and functions of postnatal tissues and metastasis of tumor cells that, like fetal hepatocytes during liver development, require actin cytoskeletal reorganization. ARF6 could also be involved in many other settings, e.g., pathogenesis of the Vibrio cholerae bacterium-induced diarrhea through the promotion of ADP-ribosylation of Gs{alpha} (10) by cholera toxin in intestinal epithelial cells. To clarify these functions, it will be necessary to generate conditional knockout mice, since ARF6-null mice invariably exhibit embryonic lethality.


   ACKNOWLEDGMENTS
 
This work was supported by research grants from the Ministry of Education, Science, Sports and Culture, Japan to Y. K. and from the Mitsubishi research foundation to Y. K.

We thank H. Nishina and K. Miyazawa for valuable comments and advice. We are also greatly appreciative to J. Penninger, J. G. Donaldson, and K. Nakayama for their generous gifts of E14K ES cells and a mouse 129/Ola genomic library, the anti-ARF6 antiserum, and ARF6 cDNA, respectively.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Physiological Chemistry, Graduate School of Comprehensive Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Ten-nohdai, Tsukuba-shi, Ibaraki 305-8575, Japan. Phone: 81-29-853-3282. Fax: 29-853-3271. E-mail: ykanaho{at}md.tsukuba.ac.jp. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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Molecular and Cellular Biology, August 2006, p. 6149-6156, Vol. 26, No. 16
0270-7306/06/$08.00+0     doi:10.1128/MCB.00298-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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